RNA interference suppression of neurodegenerative diseases and methods of use thereof

ABSTRACT

The present invention is directed to RNA interference (RNAi) molecules targeted against a nucleic acid sequence that encodes poly-glutamine repeat diseases, and methods of using these RNAi molecules.

PRIORITY OF INVENTION

This application is a continuation application of U.S. application Ser.No. 12/977,812, which was filed on Dec. 23, 2010, which is acontinuation application of U.S. application Ser. No. 12/111,025, whichwas filed on Apr. 28, 2008, which application is related to and claimspriority under 35 U.S.C. §119(e) to U.S. Provisional Application No.60/914,309 filed on Apr. 26, 2007, which are incorporated by referenceherein.

BACKGROUND OF THE INVENTION

Double-stranded RNA (dsRNA) can induce sequence-specificposttranscriptional gene silencing in many organisms by a process knownas RNA interference (RNAi). However, in mammalian cells, dsRNA that is30 base pairs or longer can induce sequence-nonspecific responses thattrigger a shut-down of protein synthesis. RNA fragments are thesequence-specific mediators of RNAi. Interference of gene expression bythese RNA interference (RNAi) molecules is now recognized as a naturallyoccurring strategy for silencing genes in the cells of many organisms.

SUMMARY OF THE INVENTION

The dominant polyglutamine expansion diseases, which includeHuntington's disease (HD), are progressive, untreatableneurodegenerative disorders. In inducible mouse models of HD, repressionof mutant allele expression improves disease phenotypes. Thus, therapiesdesigned to inhibit disease gene expression would be beneficial. Thepresent invention provides methods of using RNAi in vivo to treatdominant neurodegenerative diseases. “Treating” as used herein refers toameliorating at least one symptom of, curing and/or preventing thedevelopment of a disease or a condition.

In certain embodiment of the invention, RNAi molecules are employed toinhibit expression of a target gene. By “inhibit expression” is meant toreduce, diminish or suppress expression of a target gene. Expression ofa target gene may be inhibited via “gene silencing.” Gene silencingrefers to the suppression of gene expression, e.g., transgene,heterologous gene and/or endogenous gene expression, which may bemediated through processes that affect transcription and/or throughprocesses that affect post-transcriptional mechanisms. In someembodiments, gene silencing occurs when an RNAi molecule initiates thedegradation of the mRNA transcribed from a gene of interest in asequence-specific manner via RNA interference, thereby preventingtranslation of the gene's product.

The present invention provides an isolated RNA duplex (underphysiological conditions) comprising a first strand of RNA and a secondstrand of RNA, wherein the first strand comprises at least 15 contiguousnucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS 18 (SEQ ID NO:2),HDAS 19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), and wherein the secondstrand is complementary to at least 12 contiguous nucleotides of thefirst strand. As used herein the term “encoded by” is used in a broadsense, similar to the term “comprising” in patent terminology. Forexample, the statement “the first strand of RNA is encoded by SEQ IDNO:1” means that the first strand of RNA sequence corresponds to the RNAsequence transcribed from the DNA sequence indicated in SEQ ID NO:1, butmay also contain additional nucleotides at either the 3′ end or at the5′ end of the RNA molecule.

The reference to siRNAs herein is meant to include shRNAs and othersmall RNAs that can or are capable of modulating the expression of HDgene, for example via RNA interference. Such small RNAs include withoutlimitation, shRNAs and miroRNAs (miRNAs).

In certain embodiments, the RNA duplex described above is between 15 and30 base pairs in length, such as between 19 and 25 base pairs, such as19 or 21 base pairs in length. In certain embodiments, the first and/orsecond strand further comprises an overhang, such as a 3′ overhangregion, a 5′ overhang region, or both 3′ and 5′ overhang regions. Thetwo strands of RNA in the siRNA may be completely complementary, or oneor the other of the strands may have an “overhang region” (i.e., aportion of the RNA that does not bind with the second strand). Such anoverhang region may be from 1 to 10 nucleotides in length.

In certain embodiments, in the RNA duplex described above, the firststrand and the second strand are operably linked by means of an RNA loopstrand to form a hairpin structure to form a duplex structure and a loopstructure. In certain embodiments, the loop structure contains from 4 to50 nucleotides. In certain embodiments, the loop structure contains from4 to 10 nucleotides, such as 4, 5 or 6 nucleotides.

In certain embodiments, the loop portion is designed to circumventexportin-5 mediated export. The loop can vary in length. In someembodiments the loop is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In certainembodiments, the loop portion is a 30 nucleotide L1 motif. In certainembodiments, the loop portion is about 12 to 50 nucleotides long, or isabout 20 to 40 nucleotides long, or is about 25 to 35 nucleotides long,or is about 30 nucleotides long. In certain embodiments, the loopportion is a 32 nucleotide L1 motif. In certain embodiments, the loopportion comprises between 12 and 32 nucleotides of SEQ ID NO:9. Incertain embodiments, the loop portion comprises between 12 and 32contiguous nucleotides of SEQ ID NO:9. In certain embodiments, the loopportion consists of SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.Exemplary loop portions are provided below:

NES-long #1: (SEQ ID NO: 9) 5′-ACACAGGAAG GGGAAUAUCA CACUCUGGGG AU-3′NES-long #2: (SEQ ID NO: 11) 5′-ACACAGGAAG GGGAAUAUCA CACUCUGGGA U-3′NES-short: (SEQ ID NO: 10) 5′-ACACAGGAAG GGGAU-3′ NES-long #1:(SEQ ID NO: 12) 5′-CACAGGAAGG GGAAUAUCAC ACUCUGGGGA-3′ NES-long #2:(SEQ ID NO: 13) 5′-CACAGGAAGG GGAAUAUCAC ACUCUGGGA-3′ NES-short:(SEQ ID NO: 14) 5′-CACAGGAAGG GGA-3′

The present invention further provides expression cassettes containing anucleic acid encoding HDAS 07 (SEQ ID NO:1), HDAS 18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3), HDAS 20 (SEQ ID NO:4), miHD7A-1 (SEQ ID NO:5),miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ IDNO:8). The expression cassette may further contain a promoter, such as aregulatable promoter or a constitutive promoter. Examples of suitablepromoters include a CMV, RSV, pol II or pol III promoter. The expressioncassette may further contain a polyadenylation signal (such as asynthetic minimal polyadenylation signal) and/or a marker gene. Examplesof marker genes include visual markers such as GFP, or functionalmarkers, such as antibiotic resistance genes.

The present invention also provides vectors containing an expressioncassettes containing a nucleic acid encoding HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS 19 (SEQ ID NO:3), HDAS 20 (SEQ ID NO:4), miHD7A-1(SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), ormiHD7B-2 (SEQ ID NO:8). The present invention provides a vectorcontaining a first expression cassette and a second expression cassette,wherein the first expression cassette contains a first nucleic acidencoding at least 15 contiguous nucleotides encoded by HDAS 07 (SEQ IDNO:1), HDAS 18 (SEQ ID NO:2), HDAS 19 (SEQ ID NO:3) or HDAS 20 (SEQ IDNO:4), and the second expression cassette contains a second nucleic acidencoding at least 12 contiguous nucleotides complementary to the firststrand. Examples of appropriate vectors include adenoviral, lentiviral,adeno-associated viral (AAV), poliovirus, HSV, or murine Maloney-basedviral vectors. In one embodiment, the vector is an adenoviral vector. Incertain embodiments, a vector may contain two expression cassettes, afirst expression cassette containing a nucleic acid encoding the firststrand of the RNA duplex and a second expression cassette containing anucleic acid encoding the second strand of the RNA duplex.

The present invention provides cells (such as a mammalian cell)containing an expression cassette expression containing a nucleic acidencoding HDAS 07 (SEQ ID NO:1), HDAS 18 (SEQ ID NO:2), HDAS 19 (SEQ IDNO:3), HDAS 20 (SEQ ID NO:4), miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ IDNO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8); a vectorcontaining an expression cassettes containing a nucleic acid encodingHDAS 07 (SEQ ID NO:1), HDAS 18 (SEQ ID NO:2), HDAS 19 (SEQ ID NO:3),HDAS 20 (SEQ ID NO:4), miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6),miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8); or a vectorcontaining a first expression cassette and a second expression cassette,wherein the first expression cassette contains a first nucleic acidencoding at least 15 contiguous nucleotides encoded by HDAS 07 (SEQ IDNO:1), HDAS 18 (SEQ ID NO:2), HDAS 19 (SEQ ID NO:3) or HDAS 20 (SEQ IDNO:4), and the second expression cassette contains a second nucleic acidencoding at least 12 contiguous nucleotides complementary to the firststrand. The present invention also provides a non-human mammalcontaining these expression cassettes or vectors described herein. Incertain embodiments, the vector is an adenoviral, lentiviral,adeno-associated viral (AAV), poliovirus, HSV, or murine Maloney-basedviral vector.

The present invention provides a method of suppressing the accumulationof huntingtin in a cell by introducing a ribonucleic acid (RNA) duplexinto the cell in an amount sufficient to suppress accumulation ofhuntingtin in the cell, wherein the RNA duplex contains (a) an isolatedor purified miRNA consisting of miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ IDNO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8); (b) a firststrand of RNA and a second strand of RNA, wherein the first strandcontains at least 15 contiguous nucleotides encoded by HDAS 07 (SEQ IDNO:1), HDAS 18 (SEQ ID NO:2), HDAS 19 (SEQ ID NO:3) or HDAS 20 (SEQ IDNO:4), and wherein the second strand is complementary to at least 12contiguous nucleotides of the first strand; (c) a vector containing anexpression cassettes containing a nucleic acid encoding HDAS 07 (SEQ IDNO:1), HDAS 18 (SEQ ID NO:2), HDAS 19 (SEQ ID NO:3), HDAS 20 (SEQ IDNO:4), miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ IDNO:7), or miHD7B-2 (SEQ ID NO:8); or (d) a vector containing a firstexpression cassette and a second expression cassette, wherein the firstexpression cassette contains a first nucleic acid encoding at least 15contiguous nucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS 18 (SEQ IDNO:2), HDAS 19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), and the secondexpression cassette contains a second nucleic acid encoding at least 12contiguous nucleotides complementary to the first strand. In certainembodiments, the accumulation of huntingtin is suppressed by at least10%. The accumulation of huntingtin is suppressed by at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% 95%, or 99%.

The present invention provides a method of preventing cytotoxic effectsof mutant huntingtin in a cell by introducing a ribonucleic acid (RNA)duplex into the cell in an amount sufficient to suppress accumulation ofhuntingtin, and wherein the RNA prevents cytotoxic effects of huntingtinin the cell, wherein the RNA duplex contains (a) an isolated or purifiedmiRNA consisting of miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6),miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8); (b) a first strand ofRNA and a second strand of RNA, wherein the first strand contains atleast 15 contiguous nucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS 19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), andwherein the second strand is complementary to at least 12 contiguousnucleotides of the first strand; (c) a vector containing an expressioncassettes containing a nucleic acid encoding HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS 19 (SEQ ID NO:3), HDAS 20 (SEQ ID NO:4), miHD7A-1(SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), ormiHD7B-2 (SEQ ID NO:8); or (d) a vector containing a first expressioncassette and a second expression cassette, wherein the first expressioncassette contains a first nucleic acid encoding at least 15 contiguousnucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS 18 (SEQ ID NO:2),HDAS 19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), and the secondexpression cassette contains a second nucleic acid encoding at least 12contiguous nucleotides complementary to the first strand.

The present invention provides a method to inhibit expression of ahuntingtin gene in a cell by introducing a ribonucleic acid (RNA) intothe cell in an amount sufficient to inhibit expression of thehuntingtin, and wherein the RNA duplex contains (a) an isolated orpurified miRNA consisting of miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ IDNO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8), or (b) a firststrand of RNA and a second strand of RNA, wherein the first strandcontains at least 15 contiguous nucleotides encoded by HDAS 07 (SEQ IDNO:1), HDAS 18 (SEQ ID NO:2), HDAS 19 (SEQ ID NO:3) or HDAS 20 (SEQ IDNO:4), and wherein the second strand is complementary to at least 12contiguous nucleotides of the first strand. The huntingtin is inhibitedby at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%, or 99%.

The present invention provides a method to inhibit expression of ahuntingtin gene in a mammal (e.g., a human) by (a) providing a mammalcontaining a neuronal cell, wherein the neuronal cell contains thehuntingtin gene and the neuronal cell is susceptible to RNAinterference, and the huntingtin gene is expressed in the neuronal cell;and (b) contacting the mammal with (i) an isolated or purified miRNAconsisting of miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1(SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8); (ii) a first strand of RNA anda second strand of RNA, wherein the first strand comprises at least 15contiguous nucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS 18 (SEQ IDNO:2), HDAS 19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), and wherein thesecond strand is complementary to at least 12 contiguous nucleotides ofthe first strand; (iii) a vector comprising an expression cassettescomprising a nucleic acid encoding HDAS 07 (SEQ ID NO:1), HDAS 18 (SEQID NO:2), HDAS 19 (SEQ ID NO:3), HDAS 20 (SEQ ID NO:4), miHD7A-1 (SEQ IDNO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQID NO:8); or (iv) a vector comprising a first expression cassette and asecond expression cassette, wherein the first expression cassettecomprises a first nucleic acid encoding at least 15 contiguousnucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS 18 (SEQ ID NO:2),HDAS 19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), and the secondexpression cassette comprises a second nucleic acid encoding at least 12contiguous nucleotides complementary to the first strand. In certainembodiments, the accumulation of huntingtin is suppressed by at least10%. The huntingtin is inhibited by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% 95%, or 99%. In certain embodiments, the cell locatedin vivo in a mammal.

The present invention provides a vector comprising a promoter and amicro RNA (miRNA) shuttle containing an embedded siRNA that specificallytargets a target sequence associated with a condition amenable to siRNAtherapy, wherein the miRNA shuttle encodes (a) an isolated first strandof RNA of 15 to 30 nucleotides in length and an isolated second strandof RNA of 15 to 30 nucleotides in length, wherein the first strandcontains at least 15 contiguous nucleotides encoded by HDAS 07 (SEQ IDNO:1), HDAS 18 (SEQ ID NO:2), HDAS 19 (SEQ ID NO:3) or HDAS 20 (SEQ IDNO:4); or (b) miRNA containing miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ IDNO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8). In certainembodiments, the promoter is an inducible promoter. In certainembodiments, the vector is a viral vector. In certain embodiments, thevector is an adenoviral, lentiviral, adeno-associated viral (AAV),poliovirus, HSV, or murine Maloney-based viral vector.

The present invention provides a vector containing a first expressioncassette and a second expression cassette, wherein the first expressioncassette contains a first nucleic acid encoding at least 15 contiguousnucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS 18 (SEQ ID NO:2),HDAS 19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), and the secondexpression cassette contains a second nucleic acid encoding at least 12contiguous nucleotides complementary to the first strand.

The present invention provides a method of preventing cytotoxic effectsof neurodegenerative disease in a mammal in need thereof, by introducingthe vector encoding a miRNA described in the preceding paragraph into acell in an amount sufficient to suppress accumulation of a proteinassociated with the neurodegenerative disease, and wherein the RNAprevents cytotoxic effects of neurodegenerative disease.

The present invention also provides a method to inhibit expression of aprotein associated with the neurodegenerative disease in a mammal inneed thereof, by introducing the vector encoding a miRNA described aboveinto a cell in an amount sufficient to inhibit expression of the proteinassociated with the neurodegenerative disease, wherein the RNA inhibitsexpression of the protein associated with the neurodegenerative disease.The protein associated with the neurodegenerative disease is inhibitedby at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%, or 99%.

This invention relates to compounds, compositions, and methods usefulfor modulating Huntington's Disease (also referred to as huntingtin,htt, or HD) gene expression using short interfering nucleic acid (siRNA)molecules. This invention also relates to compounds, compositions, andmethods useful for modulating the expression and activity of other genesinvolved in pathways of HD gene expression and/or activity by RNAinterference (RNAi) using small nucleic acid molecules. In particular,the instant invention features small nucleic acid molecules, such asshort interfering nucleic acid (siRNA), short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA(shRNA) molecules and methods used to modulate the expression HD genes.A siRNA of the instant invention can be chemically synthesized,expressed from a vector or enzymatically synthesized.

In one embodiment, the present invention provides an AAV-1 expressedsiRNA comprising an isolated first strand of RNA of 15 to 30 nucleotidesin length and an isolated second strand of RNA of 15 to 30 nucleotidesin length, wherein the first strand of RNA comprises at least 15contiguous nucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS 18 (SEQ IDNO:2), HDAS 19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4). wherein the firstor second strand comprises a sequence that is complementary to anucleotide sequence encoding a mutant Huntington's Disease protein,wherein at least 12 nucleotides of the first and second strands arecomplementary to each other and form a small interfering RNA (siRNA)duplex under physiological conditions, and wherein the siRNA silencesthe expression of the nucleotide sequence encoding the mutantHuntington's Disease protein in the cell. In one embodiment, the firstor second strand comprises a sequence that is complementary to both amutant and wild-type Huntington's disease allele, and the siRNA silencesthe expression of the nucleotide sequence encoding the mutantHuntington's Disease protein and wild-type Huntington's Disease proteinin the cell. In one embodiment, an AAV-1 vector of the invention is apsuedotyped rAAV-1 vector.

The present invention provides an isolated or purified miRNA consistingof miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ IDNO:7), or miHD7B-2 (SEQ ID NO:8).

The present invention provides a mammalian cell containing an isolatedfirst strand of RNA of 15 to 30 nucleotides in length, and an isolatedsecond strand of RNA of 15 to 30 nucleotides in length, wherein thefirst strand contains a sequence that is complementary to for example atleast 15 nucleotides of RNA encoded by a targeted gene of interest (forexample the HD gene), wherein for example at least 12 nucleotides of thefirst and second strands are complementary to each other and form asmall interfering RNA (siRNA) duplex for example under physiologicalconditions, and wherein the siRNA silences (for example via RNAinterference) only one allele of the targeted gene (for example themutant allele of HD gene) in the cell. The duplex of the siRNA may bebetween 15 and 30 base pairs in length. The two strands of RNA in thesiRNA may be completely complementary, or one or the other of thestrands may have an “overhang region” or a “bulge region” (i.e., aportion of the RNA that does not bind with the second strand or where aportion of the RNA sequence is not complementary to the sequence of theother strand). These overhangs may be at the 3′ end or at the 5′ region,or at both 3′ and 5′ ends. Such overhang regions may be from 1 to 10(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) or more nucleotides in length. Thebulge regions may be at the ends or in the internal regions of the siRNAduplex. Such bulge regions may be from 1-5 (e.g., 1, 2, 3, 4, 5) or morenucleotides long. Such bulge regions may be the bulge regionscharacteristics of miRNAs. In the present invention, the first andsecond strand of RNA may be operably linked together by means of an RNAloop strand to form a hairpin structure to form a “duplex structure” anda “loop structure.” These loop structures may be from 4 to 10 (e.g., 4,5, 6, 7, 8, 9, 10) or more nucleotides in length. For example, the loopstructure may be 4, 5 or 6 nucleotides long.

The present invention also provides a mammalian cell that contains anexpression cassette encoding an isolated first strand of RNA of 15 to 30nucleotides in length, and an isolated second strand of RNA of 15 to 30nucleotides in length, wherein the first strand contains a sequence thatis complementary to for example at least 15 contiguous nucleotides ofRNA encoded by a targeted gene of interest (for example the HD gene),wherein for example at least 12 nucleotides of the first and secondstrands are complementary to each other and form a small interfering RNA(siRNA) duplex, for example under physiological conditions, and whereinthe siRNA silences (for example via RNA interference) only one allele ofthe targeted gene (for example the mutant allele of HD gene) in thecell. These expression cassettes may further contain a promoter. Suchpromoters can be regulatable promoters or constitutive promoters.Examples of suitable promoters include a CMV, RSV, pol II or pol IIIpromoter. The expression cassette may further contain a polyadenylationsignal, such as a synthetic minimal polyadenylation signal. Theexpression cassette may further contain a marker gene. The expressioncassette may be contained in a vector. Examples of appropriate vectorsinclude adenoviral, lentiviral, adeno-associated viral (AAV),poliovirus, HSV, or murine Maloney-based viral vectors. In oneembodiment, the vector is an adenoviral vector or an adeno-associatedviral vector.

In the present invention, the alleles of the targeted gene may differ byseven or fewer nucleotides (e.g., 7, 6, 5, 4, 3, 2 or 1 nucleotides).For example the alleles may differ by only one nucleotide. Examples oftargeted gene transcripts include transcripts encoding abeta-glucuronidase, TorsinA, Ataxin-3, Tau, or huntingtin. The targetedgenes and gene products (i.e., a transcript or protein) may be fromdifferent species of organisms, such as a mouse allele or a human alleleof a target gene.

The RNA duplexes of the present invention are between 15 and 30 basepairs in length. For example they may be between 19 and 25 base pairs inlength or 19-27 base-pairs in length. As discussed above the firstand/or second strand further may optionally comprise an overhang region.These overhangs may be at the 3′ end or at the 5′ overhang region, or atboth 3′ and 5′ ends. Such overhang regions may be from 1 to 10nucleotides in length. The RNA duplex of the present invention mayoptionally include nucleotide bulge regions. The bulge regions may be atthe ends or in the internal regions of the siRNA duplex. Such bulgeregions may be from 1-5 nucleotides long. Such bulge regions may be thebulge regions characteristics of miRNAs. In the present invention, thefirst and second strand of RNA may be operably linked together by meansof an RNA loop strand to form a hairpin structure to form a “duplexstructure” and a “loop structure.” These loop structures may be from 4to 10 nucleotides in length. For example, the loop structure may be 4, 5or 6 nucleotides long.

In the present invention, an expression cassette may contain a nucleicacid encoding at least one strand of the RNA duplex described above.Such an expression cassette may further contain a promoter. Theexpression cassette may be contained in a vector. These cassettes andvectors may be contained in a cell, such as a mammalian cell. Anon-human mammal may contain the cassette or vector. The vector maycontain two expression cassettes, the first expression cassettecontaining a nucleic acid encoding the first strand of the RNA duplex,and a second expression cassette containing a nucleic acid encoding thesecond strand of the RNA duplex.

In one embodiment, the present invention further provides a method ofperforming gene silencing in a mammal or mammalian cell by administeringto the mammal an isolated first strand of RNA of about 15 to about 30nucleotides (for example 19-27 nucleotides) in length, and an isolatedsecond strand of RNA of 15 to 30 nucleotides (for example 19-27nucleotides) in length, wherein the first strand contains for example atleast 15 contiguous nucleotides complementary to a targeted gene ofinterest (such as HD gene), wherein for example at least 12 nucleotidesof the first and second strands are complementary to each other and forma small interfering RNA (siRNA) duplex for example under physiologicalconditions, and wherein the siRNA silences only one or both alleles ofthe targeted gene (for example the wild type and mutant alleles of HDgene) in the mammal or mammalian cell. In one example, the gene is abeta-glucuronidase gene. The alleles may be murine-specific andhuman-specific alleles of beta-glucuronidase. Examples of genetranscripts include an RNA transcript complementary to TorsinA,Ataxin-3, huntingtin or Tau. The targeted gene may be a gene associatedwith a condition amenable to siRNA therapy. For example, the conditionamenable to siRNA therapy could be a disabling neurological disorder.

“Neurological disease” and “neurological disorder” refer to bothhereditary and sporadic conditions that are characterized by nervoussystem dysfunction, and which may be associated with atrophy of theaffected central or peripheral nervous system structures, or loss offunction without atrophy. A neurological disease or disorder thatresults in atrophy is commonly called a “neurodegenerative disease” or“neurodegenerative disorder.” Neurodegenerative diseases and disordersinclude, but are not limited to, amyotrophic lateral sclerosis (ALS),hereditary spastic hemiplegia, primary lateral sclerosis, spinalmuscular atrophy, Kennedy's disease, Alzheimer's disease, Parkinson'sdisease, multiple sclerosis, and repeat expansion neurodegenerativediseases, e.g., diseases associated with expansions of trinucleotiderepeats such as polyglutamine (polyQ) repeat diseases, e.g.,Huntington's disease (HD), spinocerebellar ataxia (SCA1, SCA2, SCA3,SCA6, SCAT, and SCA17), spinal and bulbar muscular atrophy (SBMA),dentatorubropallidoluysian atrophy (DRPLA). An example of a disablingneurological disorder that does not appear to result in atrophy is DYT1dystonia. The gene of interest may encode a ligand for a chemokineinvolved in the migration of a cancer cell, or a chemokine receptor.

The present invention further provides a method of substantiallysilencing a target gene of interest or targeted allele for the gene ofinterest in order to provide a therapeutic effect. As used herein theterm “substantially silencing” or “substantially silenced” refers todecreasing, reducing, or inhibiting the expression of the target gene ortarget allele by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% to 100%. As used herein theterm “therapeutic effect” refers to a change in the associatedabnormalities of the disease state, including pathological andbehavioral deficits; a change in the time to progression of the diseasestate; a reduction, lessening, or alteration of a symptom of thedisease; or an improvement in the quality of life of the personafflicted with the disease. Therapeutic effect can be measuredquantitatively by a physician or qualitatively by a patient afflictedwith the disease state targeted by the siRNA. In certain embodimentswherein both the mutant and wild type allele are substantially silenced,the term therapeutic effect defines a condition in which silencing ofthe wild type allele's expression does not have a deleterious or harmfuleffect on normal functions such that the patient would not have atherapeutic effect.

In one embodiment, the present invention further provides a method ofperforming allele-specific gene silencing in a mammal by administeringto the mammal an isolated first strand of RNA of 15 to 30 nucleotides inlength, and an isolated second strand of RNA of 15 to 30 nucleotides inlength, wherein the first strand contains for example at least 15contiguous nucleotides complementary to a targeted gene of interest,wherein for example at least 12 nucleotides of the first and secondstrands are complementary to each other and form a small interfering RNA(siRNA) duplex for example under physiological conditions, and whereinthe siRNA silences only one allele of the targeted gene in the mammal.The alleles of the gene may differ by seven or fewer base pairs, such asby only one base pair. In one example, the gene is a beta-glucuronidasegene. The alleles may be murine-specific and human-specific alleles ofbeta-glucuronidase. Examples of gene transcripts include an RNAtranscript complementary to TorsinA, Ataxin-3, huntingtin or Tau. Thetargeted gene may be a gene associated with a condition amenable tosiRNA therapy. For example, the condition amenable to siRNA therapycould be a disabling neurological disorder.

“Neurological disease” and “neurological disorder” refer to bothhereditary and sporadic conditions that are characterized by nervoussystem dysfunction, and which may be associated with atrophy of theaffected central or peripheral nervous system structures, or loss offunction without atrophy. A neurological disease or disorder thatresults in atrophy is commonly called a “neurodegenerative disease” or“neurodegenerative disorder.” Neurodegenerative diseases and disordersinclude, but are not limited to, amyotrophic lateral sclerosis (ALS),hereditary spastic hemiplegia, primary lateral sclerosis, spinalmuscular atrophy, Kennedy's disease, Alzheimer's disease, Parkinson'sdisease, multiple sclerosis, and repeat expansion neurodegenerativediseases, e.g., diseases associated with expansions of trinucleotiderepeats such as polyglutamine (polyQ) repeat diseases, e.g.,Huntington's disease (HD), spinocerebellar ataxia (SCA1, SCA2, SCA3,SCA6, SCA7, and SCA17), spinal and bulbar muscular atrophy (SBMA),dentatorubropallidoluysian atrophy (DRPLA). An example of a disablingneurological disorder that does not appear to result in atrophy is DYT1dystonia. The gene of interest may encode a ligand for a chemokineinvolved in the migration of a cancer cell, or a chemokine receptor.

In one embodiment, the present invention further provides a method ofsubstantially silencing both alleles (e.g., both mutant and wild typealleles) of a target gene. In certain embodiments, the targeting of bothalleles of a gene target of interest can confer a therapeutic effect byallowing a certain level of continued expression of the wild-type allelewhile at the same time inhibiting expression of the mutant (e.g.,disease associated) allele at a level that provides a therapeuticeffect. For example, a therapeutic effect can be achieved by conferringon the cell the ability to express siRNA as an expression cassette,wherein the expression cassette contains a nucleic acid encoding a smallinterfering RNA molecule (siRNA) targeted against both alleles, andwherein the expression of the targeted alleles are silenced at a levelthat inhibits, reduces, or prevents the deleterious gain of functionconferred by the mutant allele, but that still allows for adequateexpression of the wild type allele at a level that maintains thefunction of the wild type allele. Examples of such wild type and mutantalleles include without limitation those associated with polyglutaminediseases such as Huntington's Disease.

In one embodiment, the present invention further provides a method ofsubstantially silencing a target allele while allowing expression of awild-type allele by conferring on the cell the ability to express siRNAas an expression cassette, wherein the expression cassette contains anucleic acid encoding a small interfering RNA molecule (siRNA) targetedagainst a target allele, wherein expression from the targeted allele issubstantially silenced but wherein expression of the wild-type allele isnot substantially silenced.

In one embodiment, the present invention provides a method of treating adominantly inherited disease in an allele-specific manner byadministering to a patient in need thereof an expression cassette,wherein the expression cassette contains a nucleic acid encoding a smallinterfering RNA molecule (siRNA) targeted against a target allele,wherein expression from the target allele is substantially silenced butwherein expression of the wild-type allele is not substantiallysilenced.

In one embodiment, the present invention provides a method of treating adominantly inherited disease by administering to a patient in needthereof an expression cassette, wherein the expression cassette containsa nucleic acid encoding a small interfering RNA molecule (siRNA)targeted against both the mutant allele and the wild type allele of thetarget gene, wherein expression from the mutant allele is substantiallysilenced at a level that still allows for expression from the wild typeallele to maintain its function in the patient.

In one embodiment, the present invention also provides a method ofperforming allele-specific gene silencing by administering an expressioncassette containing a pol II promoter operably-linked to a nucleic acidencoding at least one strand of a small interfering RNA molecule (siRNA)targeted against a gene of interest, wherein the siRNA silences only oneallele of a gene.

In one embodiment, the present invention also provides a method ofperforming gene silencing by administering an expression cassettecontaining a pol II promoter operably-linked to a nucleic acid encodingat least one strand of a small interfering RNA molecule (siRNA) targetedagainst a gene of interest, wherein the siRNA silences one or bothalleles of the gene.

In one embodiment, the present invention provides a method of performingallele-specific gene silencing in a mammal by administering to themammal a vector containing an expression cassette, wherein theexpression cassette contains a nucleic acid encoding at least one strandof a small interfering RNA molecule (siRNA) targeted against a gene ofinterest, wherein the siRNA silences only one allele of a gene.

In one embodiment, the present invention provides a method of performinggene silencing in a mammal by administering to the mammal a vectorcontaining an expression cassette, wherein the expression cassettecontains a nucleic acid encoding at least one strand of a smallinterfering RNA molecule (siRNA) targeted against a gene of interest,wherein the siRNA silences one or both alleles of the gene.

In one embodiment, the present invention provides a method of screeningof allele-specific siRNA duplexes, involving contacting a cellcontaining a predetermined mutant allele with an siRNA with a knownsequence, contacting a cell containing a wild-type allele with an siRNAwith a known sequence, and determining if the mutant allele issubstantially silenced while the wild-type allele retains substantiallynormal activity.

In one embodiment, the present invention provides a method of screeningof specific siRNA duplexes, involving contacting a cell containing botha predetermined mutant allele and a predetermined wild-type allele withan siRNA with a known sequence, and determining if the mutant allele issubstantially silenced at a level that allows the wild-type allele toretain substantially normal activity.

In one embodiment, the present invention also provides a method ofscreening of allele-specific siRNA duplexes involving contacting a cellcontaining a predetermined mutant allele and a wild-type allele with ansiRNA with a known sequence, and determining if the mutant allele issubstantially silenced while the wild-type allele retains substantiallynormal activity.

In one embodiment, the present invention also provides a method fordetermining the function of an allele by contacting a cell containing apredetermined allele with an siRNA with a known sequence, anddetermining if the function of the allele is substantially modified.

In one embodiment, the present invention further provides a method fordetermining the function of an allele by contacting a cell containing apredetermined mutant allele and a wild-type allele with an siRNA with aknown sequence, and determining if the function of the allele issubstantially modified while the wild-type allele retains substantiallynormal function.

In one embodiment, the invention features a method for treating orpreventing Huntington's Disease in a subject or organism comprisingcontacting the subject or organism with a siRNA of the invention underconditions suitable to modulate the expression of the HD gene in thesubject or organism whereby the treatment or prevention of Huntington'sDisease can be achieved. In one embodiment, the HD gene target comprisesa mutant HD allele (e.g., an allele comprising a trinucleotide (CAG)repeat expansion). In one embodiment, the HD gene target comprises bothHD allele (e.g., an allele comprising a trinucleotide (CAG) repeatexpansion and a wild type allele). The siRNA molecule of the inventioncan be expressed from vectors as described herein or otherwise known inthe art to target appropriate tissues or cells in the subject ororganism.

In one embodiment, the invention features a method for treating orpreventing Huntington's Disease in a subject or organism comprising,contacting the subject or organism with a siRNA molecule of theinvention via local administration to relevant tissues or cells, such asbrain cells and tissues (e.g., basal ganglia, striatum, or cortex), forexample, by administration of vectors or expression cassettes of theinvention that provide siRNA molecules of the invention to relevantcells (e.g., basal ganglia, striatum, or cortex). In one embodiment, thesiRNA, vector, or expression cassette is administered to the subject ororganism by stereotactic or convection enhanced delivery to the brain.For example, U.S. Pat. No. 5,720,720 provides methods and devices usefulfor stereotactic and convection enhanced delivery of reagents to thebrain. Such methods and devices can be readily used for the delivery ofsiRNAs, vectors, or expression cassettes of the invention to a subjector organism, and is incorporated by reference herein in its entirety. USPatent Application Nos. 2002/0141980; 2002/0114780; and 2002/0187127 allprovide methods and devices useful for stereotactic and convectionenhanced delivery of reagents that can be readily adapted for deliveryof siRNAs, vectors, or expression cassettes of the invention to asubject or organism, and are incorporated by reference herein in theirentirety. Particular devices that may be useful in delivering siRNAs,vectors, or expression cassettes of the invention to a subject ororganism are for example described in US Patent Application No.2004/0162255, which is incorporated by reference herein in its entirety.The siRNA molecule of the invention can be expressed from vectors asdescribed herein or otherwise known in the art to target appropriatetissues or cells in the subject or organism.

In one embodiment, a viral vector of the invention is an AAV vector. An“AAV” vector refers to an adeno-associated virus, and may be used torefer to the naturally occurring wild-type virus itself or derivativesthereof. The term covers all subtypes, serotypes and pseudotypes, andboth naturally occurring and recombinant forms, except where requiredotherwise. As used herein, the term “serotype” refers to an AAV which isidentified by and distinguished from other AAVs based on capsid proteinreactivity with defined antisera, e.g., there are eight known serotypesof primate AAVs, AAV-1 to AAV-8. For example, serotype AAV-2 is used torefer to an AAV which contains capsid proteins encoded from the cap geneof AAV-2 and a genome containing 5′ and 3′ ITR sequences from the sameAAV-2 serotype. Pseudotyped AAV refers to an AAV that contains capsidproteins from one serotype and a viral genome including 5′-3′ ITRs of asecond serotype. Pseudotyped rAAV would be expected to have cell surfacebinding properties of the capsid serotype and genetic propertiesconsistent with the ITR serotype. Pseudotyped rAAV are produced usingstandard techniques described in the art. As used herein, for example,rAAV1 may be used to refer an AAV having both capsid proteins and 5′-3′ITRs from the same serotype or it may refer to an AAV having capsidproteins from serotype 1 and 5′-3′ ITRs from a different AAV serotype,e.g., AAV serotype 2. For each example illustrated herein thedescription of the vector design and production describes the serotypeof the capsid and 5′-3′ ITR sequences. The abbreviation “rAAV” refers torecombinant adeno-associated virus, also referred to as a recombinantAAV vector (or “rAAV vector”).

An “AAV virus” or “AAV viral particle” refers to a viral particlecomposed of at least one AAV capsid protein (preferably by all of thecapsid proteins of a wild-type AAV) and an encapsidated polynucleotide.If the particle comprises heterologous polynucleotide (i.e., apolynucleotide other than a wild-type AAV genome such as a transgene tobe delivered to a mammalian cell), it is typically referred to as“rAAV.”

In one embodiment, the AAV expression vectors are constructed usingknown techniques to at least provide as operatively linked components inthe direction of transcription, control elements including atranscriptional initiation region, the DNA of interest and atranscriptional termination region. The control elements are selected tobe functional in a mammalian cell. The resulting construct whichcontains the operatively linked components is flanked (5′ and 3′) withfunctional AAV ITR sequences.

By “adeno-associated virus inverted terminal repeats” or “AAV ITRs” ismeant the art-recognized regions found at each end of the AAV genomewhich function together in cis as origins of DNA replication and aspackaging signals for the virus. AAV ITRs, together with the AAV repcoding region, provide for the efficient excision and rescue from, andintegration of a nucleotide sequence interposed between two flankingITRs into a mammalian cell genome.

The nucleotide sequences of AAV ITR regions are known. As used herein,an “AAV ITR” need not have the wild-type nucleotide sequence depicted,but may be altered, e.g., by the insertion, deletion or substitution ofnucleotides. Additionally, the AAV ITR may be derived from any ofseveral AAV serotypes, including without limitation, AAV-1, AAV-2,AAV-3, AAV-4, AAV-5, AAVX7, etc. Furthermore, 5′ and 3′ ITRs which flanka selected nucleotide sequence in an AAV vector need not necessarily beidentical or derived from the same AAV serotype or isolate, so long asthey function as intended, i.e., to allow for excision and rescue of thesequence of interest from a host cell genome or vector, and to allowintegration of the heterologous sequence into the recipient cell genomewhen AAV Rep gene products are present in the cell.

In one embodiment, AAV ITRs can be derived from any of several AAVserotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4,AAV-5, AAVX7, etc. Furthermore, 5′ and 3′ ITRs which flank a selectednucleotide sequence in an AAV expression vector need not necessarily beidentical or derived from the same AAV serotype or isolate, so long asthey function as intended, i.e., to allow for excision and rescue of thesequence of interest from a host cell genome or vector, and to allowintegration of the DNA molecule into the recipient cell genome when AAVRep gene products are present in the cell.

In one embodiment, AAV capsids can be derived from any of several AAVserotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4,AAV-5, AAV6, or AAV8, and the AAV ITRS are derived form AAV serotype 2.Suitable DNA molecules for use in AAV vectors will be less than about 5kilobases (kb), less than about 4.5 kb, less than about 4 kb, less thanabout 3.5 kb, less than about 3 kb, less than about 2.5 kb in size. Insome embodiments of the invention the DNA molecules for use in the AAVvectors will contain multiple copies of the identical siRNA sequence. Asused herein the term multiple copies of an siRNA sequences means atleast 2 copies, at least 3 copies, at least 4 copies, at least 5 copies,at least 6 copies, at least 7 copies, at least 8 copies, at least 9copies, and at least 10 copies. In some embodiments the DNA moleculesfor use in the AAV vectors will contain multiple siRNA sequences. Asused herein the term “multiple siRNA sequences” means at least two siRNAsequences, at least three siRNA sequences, at least four siRNAsequences, at least five siRNA sequences, at least six siRNA sequences,at least seven siRNA sequences, at least eight siRNA sequences, at leastnine siRNA sequences, and at least ten siRNA sequences. In someembodiments suitable DNA vectors of the invention will contain asequence encoding the siRNA molecule of the invention and a stufferfragment. Suitable stuffer fragments of the invention include sequencesknown in the art including without limitation sequences which do notencode an expressed protein molecule; sequences which encode a normalcellular protein which would not have deleterious effect on the celltypes in which it was expressed; and sequences which would notthemselves encode a functional siRNA duplex molecule.

In one embodiment, suitable DNA molecules for use in AAV vectors will beless than about 5 kilobases (kb) in size and will include, for example,a stuffer sequence and a sequence encoding a siRNA molecule of theinvention. For example, in order to prevent any packaging of AAV genomicsequences containing the rep and cap genes, a plasmid containing the repand cap DNA fragment may be modified by the inclusion of a stufferfragment as is known in the art into the AAV genome which causes the DNAto exceed the length for optimal packaging. Thus, the helper fragment isnot packaged into AAV virions. This is a safety feature, ensuring thatonly a recombinant AAV vector genome that does not exceed optimalpackaging size is packaged into virions. An AAV helper fragment thatincorporates a stuffer sequence can exceed the wild-type genome lengthof 4.6 kb, and lengths above 105% of the wild-type will generally not bepackaged. The stuffer fragment can be derived from, for example, suchnon-viral sources as the Lac-Z or beta-galactosidase gene.

In one embodiment, the selected nucleotide sequence is operably linkedto control elements that direct the transcription or expression thereofin the subject in vivo. Such control elements can comprise controlsequences normally associated with the selected gene. Alternatively,heterologous control sequences can be employed. Useful heterologouscontrol sequences generally include those derived from sequencesencoding mammalian or viral genes. Examples include, but are not limitedto, the SV40 early promoter, mouse mammary tumor virus LTR promoter;adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV)promoter, a cytomegalovirus (CMV) promoter such as the CMV immediateearly promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, polII promoters, pol III promoters, synthetic promoters, hybrid promoters,and the like. In addition, sequences derived from nonviral genes, suchas the murine metallothionein gene, will also find use herein. Suchpromoter sequences are commercially available from, e.g., Stratagene®(San Diego, Calif.).

In one embodiment, both heterologous promoters and other controlelements, such as CNS-specific and inducible promoters, enhancers andthe like, will be of particular use. Examples of heterologous promotersinclude the CMB promoter. Examples of CNS-specific promoters includethose isolated from the genes from myelin basic protein (MBP), glialfibrillary acid protein (GFAP), and neuron specific enolase (NSE).Examples of inducible promoters include DNA responsive elements forecdysone, tetracycline, hypoxia and aufin.

In one embodiment, the AAV expression vector which harbors the DNAmolecule of interest bounded by AAV ITRs, can be constructed by directlyinserting the selected sequence(s) into an AAV genome which has had themajor AAV open reading frames (“ORFs”) excised therefrom. Other portionsof the AAV genome can also be deleted, so long as a sufficient portionof the ITRs remain to allow for replication and packaging functions.Such constructs can be designed using techniques well known in the art.See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941.

Alternatively, AAV ITRs can be excised from the viral genome or from anAAV vector containing the same and fused 5′ and 3′ of a selected nucleicacid construct that is present in another vector using standard ligationtechniques, such as those described in Sambrook and Russell, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press ColdSpring Harbor, N.Y. (2001). For example, ligations can be accomplishedin 20 mM Tris-Cl pH 7.5, 10 mM MgCl₂, 10 mM DTT, 33 μg/ml BSA, 10 mM-50mM NaCl, and either 40 μM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at0° C. (for “sticky end” ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4DNA ligase at 14° C. (for “blunt end” ligation). Intermolecular “stickyend” ligations are usually performed at 30-100 μg/ml total DNAconcentrations (5-100 nM total end concentration). AAV vectors whichcontain ITRs have been described in, e.g., U.S. Pat. No. 5,139,941. Inparticular, several AAV vectors are described therein which areavailable from the American Type Culture Collection (“ATCC”) underAccession Numbers 53222, 53223, 53224, 53225 and 53226.

Additionally, chimeric genes can be produced synthetically to includeAAV ITR sequences arranged 5′ and 3′ of one or more selected nucleicacid sequences. Preferred codons for expression of the chimeric genesequence in mammalian CNS cells can be used. The complete chimericsequence is assembled from overlapping oligonucleotides prepared bystandard methods.

In order to produce rAAV virions, an AAV expression vector is introducedinto a suitable host cell using known techniques, such as bytransfection. A number of transfection techniques are generally known inthe art. Particularly suitable transfection methods include calciumphosphate co-precipitation, direct microinjection into cultured cells,electroporation, liposome mediated gene transfer, lipid-mediatedtransduction, and nucleic acid delivery using high-velocitymicroprojectiles.

In one embodiment, suitable host cells for producing rAAV virionsinclude microorganisms, yeast cells, insect cells, and mammalian cells,that can be, or have been, used as recipients of a heterologous DNAmolecule. The term includes the progeny of the original cell which hasbeen transfected. Thus, a “host cell” as used herein generally refers toa cell which has been transfected with an exogenous DNA sequence. Cellsfrom the stable human cell line, 293 (readily available through, e.g.,the American Type Culture Collection under Accession Number ATCCCRL1573) can be used in the practice of the present invention.Particularly, the human cell line 293 is a human embryonic kidney cellline that has been transformed with adenovirus type-5 DNA fragments, andexpresses the adenoviral E1a and E1b genes. The 293 cell line is readilytransfected, and provides a particularly convenient platform in which toproduce rAAV virions.

In one embodiment, host cells containing the above-described AAVexpression vectors are rendered capable of providing AAV helperfunctions in order to replicate and encapsidate the nucleotide sequencesflanked by the AAV ITRs to produce rAAV virions. AAV helper functionsare generally AAV-derived coding sequences which can be expressed toprovide AAV gene products that, in turn, function in trans forproductive AAV replication. AAV helper functions are used herein tocomplement necessary AAV functions that are missing from the AAVexpression vectors. Thus, AAV helper functions include one, or both ofthe major AAV ORFs, namely the rep and cap coding regions, or functionalhomologues thereof.

The Rep expression products have been shown to possess many functions,including, among others: recognition, binding and nicking of the AAVorigin of DNA replication; DNA helicase activity; and modulation oftranscription from AAV (or other heterologous) promoters. The Capexpression products supply necessary packaging functions. AAV helperfunctions are used herein to complement AAV functions in trans that aremissing from AAV vectors.

The term “AAV helper construct” refers generally to a nucleic acidmolecule that includes nucleotide sequences providing AAV functionsdeleted from an AAV vector which is to be used to produce a transducingvector for delivery of a nucleotide sequence of interest. AAV helperconstructs are commonly used to provide transient expression of AAV repand/or cap genes to complement missing AAV functions that are necessaryfor lytic AAV replication; however, helper constructs lack AAV ITRs andcan neither replicate nor package themselves. AAV helper constructs canbe in the form of a plasmid, phage, transposon, cosmid, virus, orvirion. A number of AAV helper constructs have been described, such asthe commonly used plasmids pAAV/Ad and pIM29+45 which encode both Repand Cap expression products.

By “AAV rep coding region” is meant the art-recognized region of the AAVgenome which encodes the replication proteins Rep 78, Rep 68, Rep 52 andRep 40. These Rep expression products have been shown to possess manyfunctions, including recognition, binding and nicking of the AAV originof DNA replication, DNA helicase activity and modulation oftranscription from AAV (or other heterologous) promoters. The Repexpression products are collectively required for replicating the AAVgenome. Suitable homologues of the AAV rep coding region include thehuman herpesvirus 6 (HHV-6) rep gene which is also known to mediateAAV-2 DNA replication.

By “AAV cap coding region” is meant the art-recognized region of the AAVgenome which encodes the capsid proteins VP1, VP2, and VP3, orfunctional homologues thereof. These Cap expression products supply thepackaging functions which are collectively required for packaging theviral genome.

In one embodiment, AAV helper functions are introduced into the hostcell by transfecting the host cell with an AAV helper construct eitherprior to, or concurrently with, the transfection of the AAV expressionvector. AAV helper constructs are thus used to provide at leasttransient expression of AAV rep and/or cap genes to complement missingAAV functions that are necessary for productive AAV infection. AAVhelper constructs lack AAV ITRs and can neither replicate nor packagethemselves. These constructs can be in the form of a plasmid, phage,transposon, cosmid, virus, or virion. A number of AAV helper constructshave been described, such as the commonly used plasmids pAAV/Ad andpIM29+45 which encode both Rep and Cap expression products.

In one embodiment, both AAV expression vectors and AAV helper constructscan be constructed to contain one or more optional selectable markers.Suitable markers include genes which confer antibiotic resistance orsensitivity to, impart color to, or change the antigenic characteristicsof those cells which have been transfected with a nucleic acid constructcontaining the selectable marker when the cells are grown in anappropriate selective medium. Several selectable marker genes that areuseful in the practice of the invention include the hygromycin Bresistance gene (encoding Aminoglycoside phosphotranferase (APH)) thatallows selection in mammalian cells by conferring resistance to G418(available from Sigma, St. Louis, Mo.). Other suitable markers are knownto those of skill in the art.

In one embodiment, the host cell (or packaging cell) is rendered capableof providing non AAV derived functions, or “accessory functions,” inorder to produce rAAV virions. Accessory functions are non AAV derivedviral and/or cellular functions upon which AAV is dependent for itsreplication. Thus, accessory functions include at least those non AAVproteins and RNAs that are required in AAV replication, including thoseinvolved in activation of AAV gene transcription, stage specific AAVmRNA splicing, AAV DNA replication, synthesis of Cap expression productsand AAV capsid assembly. Viral-based accessory functions can be derivedfrom any of the known helper viruses.

In one embodiment, accessory functions can be introduced into and thenexpressed in host cells using methods known to those of skill in theart. Commonly, accessory functions are provided by infection of the hostcells with an unrelated helper virus. A number of suitable helperviruses are known, including adenoviruses; herpesviruses such as herpessimplex virus types 1 and 2; and vaccinia viruses. Nonviral accessoryfunctions will also find use herein, such as those provided by cellsynchronization using any of various known agents.

In one embodiment, accessory functions are provided using an accessoryfunction vector. Accessory function vectors include nucleotide sequencesthat provide one or more accessory functions. An accessory functionvector is capable of being introduced into a suitable host cell in orderto support efficient AAV virion production in the host cell. Accessoryfunction vectors can be in the form of a plasmid, phage, transposon orcosmid. Accessory vectors can also be in the form of one or morelinearized DNA or RNA fragments which, when associated with theappropriate control elements and enzymes, can be transcribed orexpressed in a host cell to provide accessory functions.

In one embodiment, nucleic acid sequences providing the accessoryfunctions can be obtained from natural sources, such as from the genomeof an adenovirus particle, or constructed using recombinant or syntheticmethods known in the art. In this regard, adenovirus-derived accessoryfunctions have been widely studied, and a number of adenovirus genesinvolved in accessory functions have been identified and partiallycharacterized. Specifically, early adenoviral gene regions E1a, E2a, E4,VAI RNA and, possibly, E1b are thought to participate in the accessoryprocess. Herpesvirus-derived accessory functions have been described.Vaccinia virus-derived accessory functions have also been described.

In one embodiment, as a consequence of the infection of the host cellwith a helper virus, or transfection of the host cell with an accessoryfunction vector, accessory functions are expressed which transactivatethe AAV helper construct to produce AAV Rep and/or Cap proteins. The Repexpression products excise the recombinant DNA (including the DNA ofinterest) from the AAV expression vector. The Rep proteins also serve toduplicate the AAV genome. The expressed Cap proteins assemble intocapsids, and the recombinant AAV genome is packaged into the capsids.Thus, productive AAV replication ensues, and the DNA is packaged intorAAV virions.

In one embodiment, following recombinant AAV replication, rAAV virionscan be purified from the host cell using a variety of conventionalpurification methods, such as CsCl gradients. Further, if infection isemployed to express the accessory functions, residual helper virus canbe inactivated, using known methods. For example, adenovirus can beinactivated by heating to temperatures of approximately 60.degrees C.for, e.g., 20 minutes or more. This treatment effectively inactivatesonly the helper virus since AAV is extremely heat stable while thehelper adenovirus is heat labile. The resulting rAAV virions are thenready for use for DNA delivery to the CNS (e.g., cranial cavity) of thesubject.

Methods of delivery of viral vectors include, but are not limited to,intra-arterial, intra-muscular, intravenous, intranasal and oral routes.Generally, rAAV virions may be introduced into cells of the CNS usingeither in vivo or in vitro transduction techniques. If transduced invitro, the desired recipient cell will be removed from the subject,transduced with rAAV virions and reintroduced into the subject.Alternatively, syngeneic or xenogeneic cells can be used where thosecells will not generate an inappropriate immune response in the subject.

Suitable methods for the delivery and introduction of transduced cellsinto a subject have been described. For example, cells can be transducedin vitro by combining recombinant AAV virions with CNS cells e.g., inappropriate media, and screening for those cells harboring the DNA ofinterest can be screened using conventional techniques such as Southernblots and/or PCR, or by using selectable markers. Transduced cells canthen be formulated into pharmaceutical compositions, described morefully below, and the composition introduced into the subject by varioustechniques, such as by grafting, intramuscular, intravenous,subcutaneous and intraperitoneal injection.

In one embodiment, for in vivo delivery, the rAAV virions are formulatedinto pharmaceutical compositions and will generally be administeredparenterally, e.g., by intramuscular injection directly into skeletal orcardiac muscle or by injection into the CNS.

In one embodiment, viral vectors of the invention are delivered to theCNS via convection-enhanced delivery (CED) systems that can efficientlydeliver viral vectors, e.g., AAV, over large regions of a subject'sbrain (e.g., striatum and/or cortex). As described in detail andexemplified below, these methods are suitable for a variety of viralvectors, for instance AAV vectors carrying therapeutic genes (e.g.,siRNAs).

Any convection-enhanced delivery device may be appropriate for deliveryof viral vectors. In one embodiment, the device is an osmotic pump or aninfusion pump. Both osmotic and infusion pumps are commerciallyavailable from a variety of suppliers, for example Alzet Corporation,Hamilton Corporation, Aiza, Inc., Palo Alto, Calif.). Typically, a viralvector is delivered via CED devices as follows. A catheter, cannula orother injection device is inserted into CNS tissue in the chosensubject. In view of the teachings herein, one of skill in the art couldreadily determine which general area of the CNS is an appropriatetarget. For example, when delivering AAV vector encoding a therapeuticgene to treat PD, the striatum is a suitable area of the brain totarget. Stereotactic maps and positioning devices are available, forexample from ASI Instruments, Warren, Mich. Positioning may also beconducted by using anatomical maps obtained by CT and/or MRI imaging ofthe subject's brain to help guide the injection device to the chosentarget. Moreover, because the methods described herein can be practicedsuch that relatively large areas of the brain take up the viral vectors,fewer infusion cannula are needed. Since surgical complications arerelated to the number of penetrations, the methods described herein alsoserve to reduce the side effects seen with conventional deliverytechniques.

In one embodiment, pharmaceutical compositions will comprise sufficientgenetic material to produce a therapeutically effective amount of thesiRNA of interest, i.e., an amount sufficient to reduce or amelioratesymptoms of the disease state in question or an amount sufficient toconfer the desired benefit. The pharmaceutical compositions will alsocontain a pharmaceutically acceptable excipient. Such excipients includeany pharmaceutical agent that does not itself induce the production ofantibodies harmful to the individual receiving the composition, andwhich may be administered without undue toxicity. Pharmaceuticallyacceptable excipients include, but are not limited to, sorbitol,Tween80, and liquids such as water, saline, glycerol and ethanol.Pharmaceutically acceptable salts can be included therein, for example,mineral acid salts such as hydrochlorides, hydrobromides, phosphates,sulfates, and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like. Additionally, auxiliarysubstances, such as wetting or emulsifying agents, pH bufferingsubstances, and the like, may be present in such vehicles. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991).

As is apparent to those skilled in the art in view of the teachings ofthis specification, an effective amount of viral vector which must beadded can be empirically determined. Administration can be effected inone dose, continuously or intermittently throughout the course oftreatment. Methods of determining the most effective means and dosagesof administration are well known to those of skill in the art and willvary with the viral vector, the composition of the therapy, the targetcells, and the subject being treated. Single and multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician.

It should be understood that more than one transgene could be expressedby the delivered viral vector. Alternatively, separate vectors, eachexpressing one or more different transgenes, can also be delivered tothe CNS as described herein. Furthermore, it is also intended that theviral vectors delivered by the methods of the present invention becombined with other suitable compositions and therapies.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B. Targeting mutant huntingtin. FIG. 1A provides aschematic representation of the huntingtin (htt) gene. The intronsequences are the lightest bands, and the exon sequences are the secondlightest bands. The expanded CAG sequence (dark band) is localized inthe first exon of the Htt gene. FIG. 1B provides siRNA walking 5′ and3′. Sequences of short interfering RNA (siRNA) targeting 5′ and 3′ ofthe CAG-repeat region were generated to preferentially target the mutanthuntingtin allele.

FIGS. 2A and 2B. Constructs to assess allele-specific silencing. Twoplasmids were generated expressing full-length wild type (FIG. 2A,pCMV-FLHtt 18Q-Flag) or mutant huntingtin (FIG. 2B, pCMV-FLHtt 83Q-V5).

FIGS. 3A-3C shows Western blots and Q-PCR results for candidate siRNAsequences. FIG. 3A shows wild type Htt and FIG. 3B shows mutant Htt. Asseen in FIG. 3C, siRNA sequence number 7 (S7) reduced mutant htt by 40%and the wild type huntingtin by 6%.

FIG. 4 shows the results of miRNA shuttles for allele-specific silencingof htt. Data represents the densitometry analysis of wild type andmutant Htt expression for different protein lysates.

FIGS. 5A and 5B. Dose response of mi7A1 sequence. FIG. 5A shows normalHtt, and FIG. 5B shows mutant Htt.

FIG. 6. Strand biasing of miR shuttles. Data represents relativeluciferase expression of the reporter constructs for each specificstrand after mil shuttle transfection. All data is compared to cellstransfected with each reporter constructs and a miRNA control (miGFP).

DETAILED DESCRIPTION OF THE INVENTION

Modulation of gene expression by endogenous, noncoding RNAs isincreasingly appreciated as a mechanism playing a role in eukaryoticdevelopment, maintenance of chromatin structure and genomic integrity.Recently, techniques have been developed to trigger RNA interference(RNAi) against specific targets in mammalian cells by introducingexogenously produced or intracellularly expressed siRNAs. These methodshave proven to be quick, inexpensive and effective for knockdownexperiments in vitro and in vivo. The ability to accomplish selectivegene silencing has led to the hypothesis that siRNAs might be employedto suppress gene expression for therapeutic benefit.

RNA interference is now established as an important biological strategyfor gene silencing, but its application to mammalian cells has beenlimited by nonspecific inhibitory effects of long double-stranded RNA ontranslation. Moreover, delivery of interfering RNA has largely beenlimited to administration of RNA molecules. Hence, such administrationmust be performed repeatedly to have any sustained effect. The presentinventors have developed a delivery mechanism that results in specificsilencing of targeted genes through expression of small interfering RNA(siRNA). The inventors have markedly diminished expression of exogenousand endogenous genes in vitro and in vivo and apply this novel strategyto a model system of a major class of neurodegenerative disorders, thepolyglutamine diseases, to show reduced polyglutamine aggregation incells. This strategy is generally useful in reducing expression oftarget genes in order to model biological processes or to providetherapy for dominant human diseases.

Disclosed herein is a strategy that results in substantial silencing oftargeted alleles via siRNA. Use of this strategy results in markedlydiminished in vitro and in vivo expression of targeted alleles. Thisstrategy is useful in reducing expression of targeted alleles in orderto model biological processes or to provide therapy for human diseases.For example, this strategy can be applied to a major class ofneurodegenerative disorders, the polyglutamine diseases, as isdemonstrated by the reduction of polyglutamine aggregation in cellsfollowing application of the strategy. As used herein the term“substantial silencing” means that the mRNA of the targeted allele isinhibited and/or degraded by the presence of the introduced siRNA, suchthat expression of the targeted allele is reduced by about 10% to 100%as compared to the level of expression seen when the siRNA is notpresent. Generally, when an allele is substantially silenced, it willhave at least 40%, 50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%,e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or even 100% reduction expression as compared to when the siRNA isnot present. As used herein the term “substantially normal activity”means the level of expression of an allele when an siRNA has not beenintroduced to a cell.

Dominantly inherited diseases, including polyQ neurodegenerativedisorders, are ideal candidates for siRNA-based therapy. The polyQneurodegenerative disorders include at least nine inherited disorderscaused by CAG repeat expansions that encode polyQ in the diseaseprotein. PolyQ expansion confers a dominant toxic property on the mutantprotein that is associated with aberrant accumulation of the diseaseprotein in neurons. All polyQ diseases are progressive, ultimately fataldisorders that typically begin in adulthood. Huntington disease (HD) isthe best known polyQ disease, but at least seven hereditary ataxias andone motor neuron disease are also due to CAG repeat/polyQ expansion.Although the clinical features and patterns of neuronal degenerationdiffer among the diseases, increasing evidence suggests that polyQdiseases share important pathogenic features. In particular, expansionof the CAG repeat/polyQ domain confers upon the encoded protein adominant toxic property. Thus as a therapeutic strategy, efforts tolower expression of the mutant gene product prior to cell death could behighly beneficial to patients.

Dominantly inherited diseases are ideal candidates for siRNA-basedtherapy. Expansions of poly-glutamine tracts in proteins that areexpressed in the central nervous system can cause neurodegenerativediseases. Some neurodegenerative diseases are caused by a (CAG)_(n)repeat that encodes poly-glutamine in a protein include Huntingtondisease (HD), spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7),spinal and bulbar muscular atrophy (SBMA), anddentatorubropallidoluysian atrophy (DRPLA). In these diseases, thepoly-glutamine expansion in a protein confers a novel toxic propertyupon the protein. Studies indicate that the toxic property is a tendencyfor the disease protein to misfold and form aggregates within neurons.Clinical characteristics of HD include progressive loss of striatalneurons and later, cortical thinning. Adult patients show choreiformmovements, impaired coordination, progressive dementia and otherpsychiatric disturbances. The symptoms of juvenile HD patients includebradykinesia, dystonia and seizures. HD is a uniformly fatal disease,with death occurring one to two decades after disease onset. In 38% ofpatients a polymorphism exists in exon 58 of the huntingtin gene,allowing for allele specific targeting.

The Hdh locus is on chromosome 4, spans 180 kb over 67 exons and encodesthe protein huntingtin (htt). In non-HD individuals, the CAG repeatregion is less than 35 CAG repeats. Expansions of 36 to ˜50 repeats, orgreater than ˜50, cause late or early onset disease, respectively. Theinverse correlation of repeat length with age of disease onset is acommon characteristic of the CAG repeat disorders, and one that isrecapitulated in mouse models. Evidence indicates that HD also may be adose-dependent process. For example, in transgenic mouse models of polyQdisease, phenotypic severity usually correlates with expression levelsof the disease protein, and homozygous transgenic mice develop diseasemore rapidly than heterozygous mice. In addition, the very rare humancases of homozygosity for polyQ disease suggest that disease severitycorrelates with the level of disease protein expression, againsupporting the notion that reducing mutant protein expression would beclinically beneficial.

The function of htt is not known. It is clear from mouse models,however, that it is required during gastrulation, neurogenesis and inpostnatal brain. Htt knock-out mice die during development. Also,removal of htt via Cre recombinase-mediated excision of a floxed Hdhallele causes progressive postnatal neurodegeneration. A CAG expansionintroduced into the mouse allele (a knock-in) does not impairneurogenesis unless wildtype htt expression is reduced from normallevels, suggesting that the expanded allele does not impair wildtype httfunction in neurogenesis. In adult mice mutant htt causes progressivedepletion of normal htt. Htt is important in vesicle trafficking, NMDAreceptor modulation, and regulation of BDNF transcription, and theexpression of many genes is affected in the CNS of HD mice.

The therapeutic promise of silencing the mutant gene (and its toxicproperty) is best demonstrated in a tetracycline-regulated mouse modelof HD. When mutant htt is inducibly expressed in these mice,pathological and behavioral features of the disease develop over time,including the characteristic formation of neuronal inclusions andabnormal motor behavior. However, when expression of the transgene isrepressed in affected mice, the pathological and behavioral features ofdisease fully resolve. This result indicates that if expression ofmutant polyQ protein can be halted, protein clearance mechanisms withinneurons can eliminate the aggregated mutant protein, and possiblynormalize mutant htt-induced changes. It also suggests that genesilencing approaches may be beneficial even for individuals with fairlyadvanced disease.

In the present invention, instead of targeting a SNP for allelespecificity, the RNAi molecules take advantage of structural integrityat the sites flanking the expansion region.

To accomplish intracellular expression of the therapeutic RNAimolecules, an RNA molecule is constructed containing two complementarystrands or a hairpin sequence (such as a 21-bp hairpin) representingsequences directed against the gene of interest. The RNAi molecule, or anucleic acid encoding the RNAi molecule, is introduced to the targetcell, such as a diseased brain cell. The RNAi molecule reduces targetmRNA and protein expression.

The construct encoding the therapeutic RNAi molecule is configured suchthat the one or more strands of the RNAi molecules are encoded by anucleic acid that is immediately contiguous to a promoter. In oneexample, the promoter is a pol II promoter. If a pol II promoter is usedin a particular construct, it is selected from readily available pol IIpromoters known in the art, depending on whether regulatable, inducible,tissue or cell-specific expression of the siRNA is desired. Theconstruct is introduced into the target cell, allowing for diminishedtarget-gene expression in the cell.

The present invention provides an expression cassette containing anisolated nucleic acid sequence encoding an RNAi molecule targetedagainst a gene of interest. The RNAi molecule may form a hairpinstructure that contains a duplex structure and a loop structure. Theloop structure may contain from 4 to 10 nucleotides, such as 4, 5 or 6nucleotides. The duplex is less than 30 nucleotides in length, such asfrom 19 to 25 nucleotides. The RNAi molecule may further contain anoverhang region. Such an overhang may be a 3′ overhang region or a 5′overhang region. The overhang region may be, for example, from 1 to 6nucleotides in length. The expression cassette may further contain a polII promoter, as described herein. Examples of pol II promoters includeregulatable promoters and constitutive promoters. For example, thepromoter may be a CMV or RSV promoter. The expression cassette mayfurther contain a polyadenylation signal, such as a synthetic minimalpolyadenylation signal. The nucleic acid sequence may further contain amarker gene or stuffer sequences. The expression cassette may becontained in a viral vector. An appropriate viral vector for use in thepresent invention may be an adenoviral, lentiviral, adeno-associatedviral (AAV), poliovirus, herpes simplex virus (HSV) or murineMaloney-based viral vector. The gene of interest may be a geneassociated with a condition amenable to siRNA therapy. Examples of suchconditions include neurodegenerative diseases, such as atrinucleotide-repeat disease (e.g., polyglutamine repeat disease).Examples of these diseases include Huntington's disease or severalspinocerebellar ataxias. Alternatively, the gene of interest may encodea ligand for a chemokine involved in the migration of a cancer cell, ora chemokine receptor.

The present invention also provides an expression cassette containing anisolated nucleic acid sequence encoding a first segment, a secondsegment located immediately 3′ of the first segment, and a third segmentlocated immediately 3′ of the second segment, wherein the first andthird segments are each less than 30 base pairs in length and each morethan 10 base pairs in length, and wherein the sequence of the thirdsegment is the complement of the sequence of the first segment, andwherein the isolated nucleic acid sequence functions as an RNAi moleculetargeted against a gene of interest. The expression cassette may becontained in a vector, such as a viral vector.

The present invention provides a method of reducing the expression of agene product in a cell by contacting a cell with an expression cassettedescribed above. It also provides a method of treating a patient byadministering to the patient a composition of the expression cassettedescribed above.

The present invention further provides a method of reducing theexpression of a gene product in a cell by contacting a cell with anexpression cassette containing an isolated nucleic acid sequenceencoding a first segment, a second segment located immediately 3′ of thefirst segment, and a third segment located immediately 3′ of the secondsegment, wherein the first and third segments are each less than 30 basepairs in length and each more than 10 base pairs in length, and whereinthe sequence of the third segment is the complement of the sequence ofthe first segment, and wherein the isolated nucleic acid sequencefunctions as an RNAi molecule targeted against a gene of interest.

The present method also provides a method of treating a patient, byadministering to the patient a composition containing an expressioncassette, wherein the expression cassette contains an isolated nucleicacid sequence encoding a first segment, a second segment locatedimmediately 3′ of the first segment, and a third segment locatedimmediately 3′ of the second segment, wherein the first and thirdsegments are each less than 30 bases in length and each more than 10bases in length, and wherein the sequence of the third segment is thecomplement of the sequence of the first segment, and wherein theisolated nucleic acid sequence functions as an RNAi molecule targetedagainst a gene of interest.

I. RNA Interference (RNAi) Molecule

An RNAi molecule may be a “small interfering RNA” or “short interferingRNA” or “siRNA” or “short hairpin RNA” or “shRNA” or “microRNA” or“miRNA.” An RNAi molecule an RNA duplex of nucleotides that is targetedto a nucleic acid sequence of interest, for example, ataxin-1 orhuntingtin (htt). As used herein, the term “RNAi molecule” is a genericterm that encompasses the subset of shRNAs. A “RNA duplex” refers to thestructure formed by the complementary pairing between two regions of aRNA molecule. RNAi molecule is “targeted” to a gene in that thenucleotide sequence of the duplex portion of the RNAi molecule iscomplementary to a nucleotide sequence of the targeted gene. In certainembodiments, the RNAi molecules are targeted to the sequence encodinghuntingtin. In some embodiments, the length of the duplex of RNAimolecules is less than 30 base pairs. In some embodiments, the duplexcan be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14,13, 12, 11 or 10 base pairs in length. In some embodiments, the lengthof the duplex is 19 to 25 base pairs in length. In certain embodiment,the length of the duplex is 19 or 21 base pairs in length. The RNAduplex portion of the RNAi molecule can be part of a hairpin structure.In addition to the duplex portion, the hairpin structure may contain aloop portion positioned between the two sequences that form the duplex.The loop can vary in length. In some embodiments the loop is 5, 6, 7, 8,9, 10, 11, 12 or 13 nucleotides in length. In certain embodiments, theloop is 9 nucleotides in length. The hairpin structure can also contain3′ or 5′ overhang portions. In some embodiments, the overhang is a 3′ ora 5′ overhang 0, 1, 2, 3, 4 or 5 nucleotides in length.

The RNAi molecule can be encoded by a nucleic acid sequence, and thenucleic acid sequence can also include a promoter. The nucleic acidsequence can also include a polyadenylation signal. In some embodiments,the polyadenylation signal is a synthetic minimal polyadenylationsignal.

“Knock-down,” “knock-down technology” refers to a technique of genesilencing in which the expression of a target gene is reduced ascompared to the gene expression prior to the introduction of the RNAimolecule, which can lead to the inhibition of production of the targetgene product. The term “reduced” is used herein to indicate that thetarget gene expression is lowered by 1-100%. In other words, the amountof RNA available for translation into a polypeptide or protein isminimized. For example, the amount of protein may be reduced by 10, 20,30, 40, 50, 60, 70, 80, 90, 95, or 99%. In some embodiments, theexpression is reduced by about 90% (i.e., only about 10% of the amountof protein is observed a cell as compared to a cell where RNAi moleculeshave not been administered). Knock-down of gene expression can bedirected, for example, by the use of dsRNAs, siRNAs or miRNAs.

“RNA interference (RNAi)” is the process of sequence-specific,post-transcriptional gene silencing initiated by an RNAi molecule.During RNAi, RNAi molecules induce degradation of target mRNA withconsequent sequence-specific inhibition of gene expression. RNAiinvolving the use of RNAi molecules has been successfully applied toknockdown the expression of specific genes in plants, D. melanogaster,C. elegans, trypanosomes, planaria, hydra, and several vertebratespecies including the mouse.

According to a method of the present invention, the expression ofhuntingtin can be modified via RNAi. For example, the accumulation ofhuntingtin can be suppressed in a cell. The term “suppressing” refers tothe diminution, reduction or elimination in the number or amount oftranscripts present in a particular cell. For example, the accumulationof mRNA encoding huntingtin can be suppressed in a cell by RNAinterference (RNAi), e.g., the gene is silenced by sequence-specificdouble-stranded RNA (dsRNA), which is also called short interfering RNA(siRNA). These siRNAs can be two separate RNA molecules that havehybridized together, or they may be a single hairpin wherein twoportions of a RNA molecule have hybridized together to form a duplex.

A mutant protein refers to the protein encoded by a gene having amutation, e.g., a missense or nonsense mutation in one or both allelesof huntingtin. A mutant huntingtin may be disease-causing, i.e., maylead to a disease associated with the presence of huntingtin in ananimal having either one or two mutant allele(s).

The term “gene” is used broadly to refer to any segment of nucleic acidassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.For example, “gene” refers to a nucleic acid fragment that expressesmRNA, functional RNA, or specific protein, including regulatorysequences. “Genes” also include nonexpressed DNA segments that, forexample, form recognition sequences for other proteins. “Genes” can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters. An“allele” is one of several alternative forms of a gene occupying a givenlocus on a chromosome.

The term “nucleic acid” refers to deoxyribonucleic acid (DNA) orribonucleic acid (RNA) and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base that is either a purine or pyrimidine.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides that have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences, as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues. A “nucleic acid fragment” is a portion of a givennucleic acid molecule.

A “nucleotide sequence” is a polymer of DNA or RNA that can be single-or double-stranded, optionally containing synthetic, non-natural oraltered nucleotide bases capable of incorporation into DNA or RNApolymers.

The terms “nucleic acid,” “nucleic acid molecule,” “nucleic acidfragment,” “nucleic acid sequence or segment,” or “polynucleotide” areused interchangeably and may also be used interchangeably with gene,cDNA, DNA and RNA encoded by a gene.

The invention encompasses isolated or substantially purified nucleicacid compositions. In the context of the present invention, an“isolated” or “purified” DNA molecule or RNA molecule is a DNA moleculeor RNA molecule that exists apart from its native environment and istherefore not a product of nature. An isolated DNA molecule or RNAmolecule may exist in a purified form or may exist in a non-nativeenvironment such as, for example, a transgenic host cell. For example,an “isolated” or “purified” nucleic acid molecule or biologically activeportion thereof, is substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized. In one embodiment, an “isolated” nucleic acid is free ofsequences that naturally flank the nucleic acid (i.e., sequences locatedat the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of theorganism from which the nucleic acid is derived. For example, in variousembodiments, the isolated nucleic acid molecule can contain less thanabout 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequences that naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. Fragments andvariants of the disclosed nucleotide sequences are also encompassed bythe present invention. By “fragment” or “portion” is meant a full lengthor less than full length of the nucleotide sequence.

“Naturally occurring,” “native,” or “wild-type” is used to describe anobject that can be found in nature as distinct from being artificiallyproduced. For example, a protein or nucleotide sequence present in anorganism (including a virus), which can be isolated from a source innature and that has not been intentionally modified by a person in thelaboratory, is naturally occurring.

A “variant” of a molecule is a sequence that is substantially similar tothe sequence of the native molecule. For nucleotide sequences, variantsinclude those sequences that, because of the degeneracy of the geneticcode, encode the identical amino acid sequence of the native protein.Naturally occurring allelic variants such as these can be identifiedwith the use of molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques. Variantnucleotide sequences also include synthetically derived nucleotidesequences, such as those generated, for example, by using site-directedmutagenesis, which encode the native protein, as well as those thatencode a polypeptide having amino acid substitutions. Generally,nucleotide sequence variants of the invention will have at least 40%,50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%,generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequenceidentity to the native (endogenous) nucleotide sequence.

The term “chimeric” refers to a gene or DNA that contains 1) DNAsequences, including regulatory and coding sequences that are not foundtogether in nature or 2) sequences encoding parts of proteins notnaturally adjoined, or 3) parts of promoters that are not naturallyadjoined. Accordingly, a chimeric gene may include regulatory sequencesand coding sequences that are derived from different sources, or includeregulatory sequences and coding sequences derived from the same source,but arranged in a manner different from that found in nature.

A “transgene” refers to a gene that has been introduced into the genomeby transformation. Transgenes include, for example, DNA that is eitherheterologous or homologous to the DNA of a particular cell to betransformed. Additionally, transgenes may include native genes insertedinto a non-native organism, or chimeric genes.

The term “endogenous gene” refers to a native gene in its naturallocation in the genome of an organism.

A “foreign” gene refers to a gene not normally found in the hostorganism that has been introduced by gene transfer.

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein.

“Conservatively modified variations” of a particular nucleic acidsequence refers to those nucleic acid sequences that encode identical oressentially identical amino acid sequences. Because of the degeneracy ofthe genetic code, a large number of functionally identical nucleic acidsencode any given polypeptide. For instance, the codons CGT, CGC, CGA,CGG, AGA and AGG all encode the amino acid arginine. Thus, at everyposition where an arginine is specified by a codon, the codon can bealtered to any of the corresponding codons described without alteringthe encoded protein. Such nucleic acid variations are “silentvariations,” which are one species of “conservatively modifiedvariations.” Every nucleic acid sequence described herein that encodes apolypeptide also describes every possible silent variation, except whereotherwise noted. One of skill in the art will recognize that each codonin a nucleic acid (except ATG, which is ordinarily the only codon formethionine) can be modified to yield a functionally identical moleculeby standard techniques. Accordingly, each “silent variation” of anucleic acid that encodes a polypeptide is implicit in each describedsequence.

“Recombinant DNA molecule” is a combination of DNA sequences that arejoined together using recombinant DNA technology and procedures used tojoin together DNA sequences as described, for example, in Sambrook andRussell (2001).

The terms “heterologous gene,” “heterologous DNA sequence,” “exogenousDNA sequence,” “heterologous RNA sequence,” “exogenous RNA sequence” or“heterologous nucleic acid” each refer to a sequence that eitheroriginates from a source foreign to the particular host cell, or is fromthe same source but is modified from its original or native form. Thus,a heterologous gene in a host cell includes a gene that is endogenous tothe particular host cell but has been modified through, for example, theuse of DNA shuffling. The terms also include non-naturally occurringmultiple copies of a naturally occurring DNA or RNA sequence. Thus, theterms refer to a DNA or RNA segment that is foreign or heterologous tothe cell, or homologous to the cell but in a position within the hostcell nucleic acid in which the element is not ordinarily found.Exogenous DNA segments are expressed to yield exogenous polypeptides.

A “homologous” DNA or RNA sequence is a sequence that is naturallyassociated with a host cell into which it is introduced.

“Wild-type” refers to the normal gene or organism found in nature.

“Genome” refers to the complete genetic material of an organism.

A “vector” is defined to include, inter alia, any viral vector, as wellas any plasmid, cosmid, phage or binary vector in double or singlestranded linear or circular form that may or may not be selftransmissible or mobilizable, and that can transform prokaryotic oreukaryotic host either by integration into the cellular genome or existextrachromosomally (e.g., autonomous replicating plasmid with an originof replication).

“Expression cassette” as used herein means a nucleic acid sequencecapable of directing expression of a particular nucleotide sequence inan appropriate host cell, which may include a promoter operably linkedto the nucleotide sequence of interest that may be operably linked totermination signals. The coding region usually codes for a functionalRNA of interest, for example an RNAi molecule. The expression cassetteincluding the nucleotide sequence of interest may be chimeric. Theexpression cassette may also be one that is naturally occurring but hasbeen obtained in a recombinant form useful for heterologous expression.The expression of the nucleotide sequence in the expression cassette maybe under the control of a constitutive promoter or of a regulatablepromoter that initiates transcription only when the host cell is exposedto some particular stimulus. In the case of a multicellular organism,the promoter can also be specific to a particular tissue or organ orstage of development.

Such expression cassettes can include a transcriptional initiationregion linked to a nucleotide sequence of interest. Such an expressioncassette is provided with a plurality of restriction sites for insertionof the gene of interest to be under the transcriptional regulation ofthe regulatory regions. The expression cassette may additionally containselectable marker genes.

“Coding sequence” refers to a DNA or RNA sequence that codes for aspecific amino acid sequence. It may constitute an “uninterrupted codingsequence”, i.e., lacking an intron, such as in a cDNA, or it may includeone or more introns bounded by appropriate splice junctions. An “intron”is a sequence of RNA that is contained in the primary transcript but isremoved through cleavage and re-ligation of the RNA within the cell tocreate the mature mRNA that can be translated into a protein.

The term “open reading frame” (ORF) refers to the sequence betweentranslation initiation and termination codons of a coding sequence. Theterms “initiation codon” and “termination codon” refer to a unit ofthree adjacent nucleotides (a ‘codon’) in a coding sequence thatspecifies initiation and chain termination, respectively, of proteinsynthesis (mRNA translation).

“Functional RNA” refers to sense RNA, antisense RNA, ribozyme RNA,siRNA, or other RNA that may not be translated but yet has an effect onat least one cellular process.

The term “RNA transcript” or “transcript” refers to the productresulting from RNA polymerase catalyzed transcription of a DNA sequence.When the RNA transcript is a perfect complementary copy of the DNAsequence, it is referred to as the primary transcript or it may be a RNAsequence derived from posttranscriptional processing of the primarytranscript and is referred to as the mature RNA. “Messenger RNA” (mRNA)refers to the RNA that is without introns and that can be translatedinto protein by the cell. “cDNA” refers to a single- or adouble-stranded DNA that is complementary to and derived from mRNA.

“Regulatory sequences” are nucleotide sequences located upstream (5′non-coding sequences), within, or downstream (3′ non-coding sequences)of a coding sequence, and which influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence. Regulatory sequences include enhancers, promoters, translationleader sequences, introns, and polyadenylation signal sequences. Theyinclude natural and synthetic sequences as well as sequences that may bea combination of synthetic and natural sequences. As is noted above, theterm “suitable regulatory sequences” is not limited to promoters.However, some suitable regulatory sequences useful in the presentinvention will include, but are not limited to constitutive promoters,tissue-specific promoters, development-specific promoters, regulatablepromoters and viral promoters.

“5′ non-coding sequence” refers to a nucleotide sequence located 5′(upstream) to the coding sequence. It is present in the fully processedmRNA upstream of the initiation codon and may affect processing of theprimary transcript to mRNA, mRNA stability or translation efficiency.

“3′ non-coding sequence” refers to nucleotide sequences located 3′(downstream) to a coding sequence and may include polyadenylation signalsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor.

The term “translation leader sequence” refers to that DNA sequenceportion of a gene between the promoter and coding sequence that istranscribed into RNA and is present in the fully processed mRNA upstream(5′) of the translation start codon. The translation leader sequence mayaffect processing of the primary transcript to mRNA, mRNA stability ortranslation efficiency.

The term “mature” protein refers to a post-translationally processedpolypeptide without its signal peptide. “Precursor” protein refers tothe primary product of translation of an mRNA. “Signal peptide” refersto the amino terminal extension of a polypeptide, which is translated inconjunction with the polypeptide forming a precursor peptide and whichis required for its entrance into the secretory pathway. The term“signal sequence” refers to a nucleotide sequence that encodes thesignal peptide.

“Promoter” refers to a nucleotide sequence, usually upstream (5′) to itscoding sequence, which directs and/or controls the expression of thecoding sequence by providing the recognition for RNA polymerase andother factors required for proper transcription. “Promoter” includes aminimal promoter that is a short DNA sequence comprised of a TATA-boxand other sequences that serve to specify the site of transcriptioninitiation, to which regulatory elements are added for control ofexpression. “Promoter” also refers to a nucleotide sequence thatincludes a minimal promoter plus regulatory elements that is capable ofcontrolling the expression of a coding sequence or functional RNA. Thistype of promoter sequence consists of proximal and more distal upstreamelements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence that can stimulate promoteractivity and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue specificity of apromoter. It is capable of operating in both orientations (normal orflipped), and is capable of functioning even when moved either upstreamor downstream from the promoter. Both enhancers and other upstreampromoter elements bind sequence-specific DNA-binding proteins thatmediate their effects. Promoters may be derived in their entirety from anative gene, or be composed of different elements derived from differentpromoters found in nature, or even be comprised of synthetic DNAsegments. A promoter may also contain DNA sequences that are involved inthe binding of protein factors that control the effectiveness oftranscription initiation in response to physiological or developmentalconditions. Examples of promoters that may be used in the presentinvention include the mouse U6 RNA promoters, synthetic human H1RNApromoters, SV40, CMV, RSV, RNA polymerase II and RNA polymerase IIIpromoters.

The “initiation site” is the position surrounding the first nucleotidethat is part of the transcribed sequence, which is also defined asposition +1. With respect to this site all other sequences of the geneand its controlling regions are numbered. Downstream sequences (i.e.,further protein encoding sequences in the 3′ direction) are denominatedpositive, while upstream sequences (mostly of the controlling regions inthe 5′ direction) are denominated negative.

Promoter elements, particularly a TATA element, that are inactive orthat have greatly reduced promoter activity in the absence of upstreamactivation are referred to as “minimal or core promoters.” In thepresence of a suitable transcription factor, the minimal promoterfunctions to permit transcription. A “minimal or core promoter” thusconsists only of all basal elements needed for transcription initiation,e.g., a TATA box and/or an initiator.

“Constitutive expression” refers to expression using a constitutive orregulated promoter. “Conditional” and “regulated expression” refer toexpression controlled by a regulated promoter.

“Operably-linked” refers to the association of nucleic acid sequences onsingle nucleic acid fragment so that the function of one of thesequences is affected by another. For example, a regulatory DNA sequenceis said to be “operably linked to” or “associated with” a DNA sequencethat codes for an RNA or a polypeptide if the two sequences are situatedsuch that the regulatory DNA sequence affects expression of the codingDNA sequence (i.e., that the coding sequence or functional RNA is underthe transcriptional control of the promoter). Coding sequences can beoperably-linked to regulatory sequences in sense or antisenseorientation.

“Expression” refers to the transcription and/or translation of anendogenous gene, heterologous gene or nucleic acid segment, or atransgene in cells. For example, in the case of siRNA constructs,expression may refer to the transcription of the siRNA only. Inaddition, expression refers to the transcription and stable accumulationof sense (mRNA) or functional RNA. Expression may also refer to theproduction of protein.

“Altered levels” refers to the level of expression in transgenic cellsor organisms that differs from that of normal or untransformed cells ororganisms.

“Overexpression” refers to the level of expression in transgenic cellsor organisms that exceeds levels of expression in normal oruntransformed cells or organisms.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of protein from anendogenous gene or a transgene.

“Transcription stop fragment” refers to nucleotide sequences thatcontain one or more regulatory signals, such as polyadenylation signalsequences, capable of terminating transcription. Examples include the 3′non-regulatory regions of genes encoding nopaline synthase and the smallsubunit of ribulose bisphosphate carboxylase.

“Translation stop fragment” refers to nucleotide sequences that containone or more regulatory signals, such as one or more termination codonsin all three frames, capable of terminating translation. Insertion of atranslation stop fragment adjacent to or near the initiation codon atthe 5′ end of the coding sequence will result in no translation orimproper translation. Excision of the translation stop fragment bysite-specific recombination will leave a site-specific sequence in thecoding sequence that does not interfere with proper translation usingthe initiation codon.

The terms “cis-acting sequence” and “cis-acting element” refer to DNA orRNA sequences whose functions require them to be on the same molecule.An example of a cis-acting sequence on the replicon is the viralreplication origin.

The terms “trans-acting sequence” and “trans-acting element” refer toDNA or RNA sequences whose function does not require them to be on thesame molecule.

“Chromosomally-integrated” refers to the integration of a foreign geneor nucleic acid construct into the host DNA by covalent bonds. Wheregenes are not “chromosomally integrated” they may be “transientlyexpressed.” Transient expression of a gene refers to the expression of agene that is not integrated into the host chromosome but functionsindependently, either as part of an autonomously replicating plasmid orexpression cassette, for example, or as part of another biologicalsystem such as a virus.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence,” (b) “comparison window,” (c) “sequence identity,” (d)“percentage of sequence identity,” and (e) “substantial identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well-known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold. These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when thecumulative alignment score falls off by the quantity X from its maximumachieved value, the cumulative score goes to zero or below due to theaccumulation of one or more negative-scoring residue alignments, or theend of either sequence is reached.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. One measure of similarity provided by the BLAST algorithmis the smallest sum probability (P(N)), which provides an indication ofthe probability by which a match between two nucleotide sequences wouldoccur by chance. For example, a test nucleic acid sequence is consideredsimilar to a reference sequence if the smallest sum probability in acomparison of the test nucleic acid sequence to the reference nucleicacid sequence is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized. Alternatively, PSI-BLAST (in BLAST 2.0) canbe used to perform an iterated search that detects distant relationshipsbetween molecules. When utilizing BLAST, Gapped BLAST, PSI-BLAST, thedefault parameters of the respective programs (e.g. BLASTN fornucleotide sequences) can be used. The BLASTN program (for nucleotidesequences) uses as defaults a wordlength (W) of 11, an expectation (E)of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands.Alignment may also be performed manually by inspection.

For purposes of the present invention, comparison of nucleotidesequences for determination of percent sequence identity to the promotersequences disclosed herein is preferably made using the BlastN program(version 1.4.7 or later) with its default parameters or any equivalentprogram. By “equivalent program” is intended any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide matches and an identical percent sequenceidentity when compared to the corresponding alignment generated by thepreferred program.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid sequences makes reference to a specified percentage ofnucleotides in the two sequences that are the same when aligned formaximum correspondence over a specified comparison window, as measuredby sequence comparison algorithms or by visual inspection.

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%,91%, 92%, 93%, or 94%, and most preferably at least 95%, 96%, 97%, 98%,or 99% sequence identity, compared to a reference sequence using one ofthe alignment programs described using standard parameters.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. However, stringent conditions encompasstemperatures in the range of about 1° C. to about 20° C., depending uponthe desired degree of stringency as otherwise qualified herein.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

As noted above, another indication that two nucleic acid sequences aresubstantially identical is that the two molecules hybridize to eachother under stringent conditions. The phrase “hybridizing specificallyto” refers to the binding, duplexing, or hybridizing of a molecule onlyto a particular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetnucleic acid sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Specificity istypically the function of post-hybridization washes, the criticalfactors being the ionic strength and temperature of the final washsolution. For DNA-DNA hybrids, the T_(m) can be approximated from theequation of Meinkoth and Wahl:T _(m)81.5° C.+16.6(log M)+0.41(% GC)−0.61(% form)−500/L;where M is the molarity of monovalent cations, % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % form is the percentageof formamide in the hybridization solution, and L is the length of thehybrid in base pairs. T_(m) is reduced by about 1° C. for each 1% ofmismatching; thus, T_(m), hybridization, and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with >90% identity are sought, the T_(m) can be decreased10° C. Generally, stringent conditions are selected to be about 5° C.lower than the thermal melting point (T_(m)) for the specific sequenceand its complement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the T_(m); moderately stringent conditions canutilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower thanthe T_(m); low stringency conditions can utilize a hybridization and/orwash at 11, 12, 13, 14, 15, or 20° C. lower than the T_(m). Using theequation, hybridization and wash compositions, and desired T, those ofordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a T of less than 45° C.(aqueous solution) or 32° C. (formamide solution), it is preferred toincrease the SSC concentration so that a higher temperature can be used.Generally, highly stringent hybridization and wash conditions areselected to be about 5° C. lower than the T_(m) for the specificsequence at a defined ionic strength and pH.

An example of highly stringent wash conditions is 0.15 M NaCl at 72° C.for about 15 minutes. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook and Russell 2001,for a description of SSC buffer). Often, a high stringency wash ispreceded by a low stringency wash to remove background probe signal. Forshort nucleic acid sequences (e.g., about 10 to 50 nucleotides),stringent conditions typically involve salt concentrations of less thanabout 1.5 M, more preferably about 0.01 to 1.0 M, Na ion concentration(or other salts) at pH 7.0 to 8.3, and the temperature is typically atleast about 30° C. Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. In general, a signalto noise ratio of 2× (or higher) than that observed for an unrelatedprobe in the particular hybridization assay indicates detection of aspecific hybridization. Very stringent conditions are selected to beequal to the Tm for a particular nucleic acid molecule.

Very stringent conditions are selected to be equal to the T_(m) for aparticular probe. An example of stringent conditions for hybridizationof complementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formamide,e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.1×SSC at 60 to 65° C. Exemplary low stringency conditionsinclude hybridization with a buffer solution of 30 to 35% formamide, 1MNaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and a wash in 1× to2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C.Exemplary moderate stringency conditions include hybridization in 40 to45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSCat 55 to 60° C.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell, resulting in genetically stableinheritance. A “host cell” is a cell that has been transformed, or iscapable of transformation, by an exogenous nucleic acid molecule. Hostcells containing the transformed nucleic acid fragments are referred toas “transgenic” cells.

“Transformed,” “transduced,” “transgenic” and “recombinant” refer to ahost cell into which a heterologous nucleic acid molecule has beenintroduced. As used herein the term “transfection” refers to thedelivery of DNA into eukaryotic (e.g., mammalian) cells. The term“transformation” is used herein to refer to delivery of DNA intoprokaryotic (e.g., E. coli) cells. The term “transduction” is usedherein to refer to infecting cells with viral particles. The nucleicacid molecule can be stably integrated into the genome generally knownin the art. Known methods of PCR include, but are not limited to,methods using paired primers, nested primers, single specific primers,degenerate primers, gene-specific primers, vector-specific primers,partially mismatched primers, and the like. For example, “transformed,”“transformant,” and “transgenic” cells have been through thetransformation process and contain a foreign gene integrated into theirchromosome. The term “untransformed” refers to normal cells that havenot been through the transformation process.

“Genetically altered cells” denotes cells which have been modified bythe introduction of recombinant or heterologous nucleic acids (e.g., oneor more DNA constructs or their RNA counterparts) and further includesthe progeny of such cells which retain part or all of such geneticmodification.

As used herein, the term “derived” or “directed to” with respect to anucleotide molecule means that the molecule has complementary sequenceidentity to a particular molecule of interest.

“Gene silencing” refers to the suppression of gene expression, e.g.,transgene, heterologous gene and/or endogenous gene expression. Genesilencing may be mediated through processes that affect transcriptionand/or through processes that affect post-transcriptional mechanisms. Insome embodiments, gene silencing occurs when siRNA initiates thedegradation of the mRNA of a gene of interest in a sequence-specificmanner via RNA interference. In some embodiments, gene silencing may beallele-specific. “Allele-specific” gene silencing refers to the specificsilencing of one allele of a gene.

“Knock-down,” “knock-down technology” refers to a technique of genesilencing in which the expression of a target gene is reduced ascompared to the gene expression prior to the introduction of the RNAimolecule, which can lead to the inhibition of production of the targetgene product. The term “reduced” is used herein to indicate that thetarget gene expression is lowered by 1-100%. For example, the expressionmay be reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or even 99%.Knock-down of gene expression can be directed by the use of dsRNAs orsiRNAs. For example, “RNA interference (RNAi),” which can involve theuse of siRNA, has been successfully applied to knockdown the expressionof specific genes in plants, D. melanogaster, C. elegans, trypanosomes,planaria, hydra, and several vertebrate species including the mouse.

“RNA interference (RNAi)” is the process of sequence-specific,post-transcriptional gene silencing initiated by siRNA. RNAi is seen ina number of organisms such as Drosophila, nematodes, fungi and plants,and is believed to be involved in anti-viral defense, modulation oftransposon activity, and regulation of gene expression. During RNAi,RNAi molecules induce degradation of target mRNA with consequentsequence-specific inhibition of gene expression.

A “small interfering” or “short interfering RNA” or siRNA is a RNAduplex of nucleotides that is targeted to a gene interest. A “RNAduplex” refers to the structure formed by the complementary pairingbetween two regions of a RNA molecule. siRNA is “targeted” to a gene inthat the nucleotide sequence of the duplex portion of the siRNA iscomplementary to a nucleotide sequence of the targeted gene. In someembodiments, the length of the duplex of siRNAs is less than 30nucleotides. In some embodiments, the duplex can be 29, 28, 27, 26, 25,24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotidesin length. In some embodiments, the length of the duplex is 19-25nucleotides in length. The RNA duplex portion of the siRNA can be partof a hairpin structure. In addition to the duplex portion, the hairpinstructure may contain a loop portion positioned between the twosequences that form the duplex. The loop can vary in length. In someembodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides inlength. The hairpin structure can also contain 3′ or 5′ overhangportions. In some embodiments, the overhang is a 3′ or a 5′ overhang 0,1, 2, 3, 4 or 5 nucleotides in length. The “sense” and “antisense”sequences can be used with or without a loop region to form siRNAmolecules. As used herein, the term siRNA is meant to be equivalent toother terms used to describe nucleic acid molecules that are capable ofmediating sequence specific RNAi, for example, double-stranded RNA(dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interferingoligonucleotide, short interfering nucleic acid, post-transcriptionalgene silencing RNA (ptgsRNA), and others. In addition, as used herein,the term RNAi is meant to be equivalent to other terms used to describesequence specific RNA interference, such as post transcriptional genesilencing, translational inhibition, or epigenetic silencing. Forexample, siRNA molecules of the invention can be used to epigeneticallysilence genes at both the post-transcriptional level or thepre-transcriptional level. In a non-limiting example, epigeneticmodulation of gene expression by siRNA molecules of the invention canresult from siRNA mediated modification of chromatin structure ormethylation pattern to alter gene expression. In another non-limitingexample, modulation of gene expression by siRNA molecules of theinvention can result from siRNA mediated cleavage of RNA (either codingor non-coding RNA) via RISC, or alternately, translational inhibition asis known in the art.

The siRNA can be encoded by a nucleic acid sequence, and the nucleicacid sequence can also include a promoter. The nucleic acid sequence canalso include a polyadenylation signal. In some embodiments, thepolyadenylation signal is a synthetic minimal polyadenylation signal.

“Treating” as used herein refers to ameliorating at least one symptomof, curing and/or preventing the development of a disease or acondition.

“Neurological disease” and “neurological disorder” refer to bothhereditary and sporadic conditions that are characterized by nervoussystem dysfunction, and which may be associated with atrophy of theaffected central or peripheral nervous system structures, or loss offunction without atrophy. A neurological disease or disorder thatresults in atrophy is commonly called a “neurodegenerative disease” or“neurodegenerative disorder.” Neurodegenerative diseases and disordersinclude, but are not limited to, amyotrophic lateral sclerosis (ALS),hereditary spastic hemiplegia, primary lateral sclerosis, spinalmuscular atrophy, Kennedy's disease, Alzheimer's disease, Parkinson'sdisease, multiple sclerosis, and repeat expansion neurodegenerativediseases, e.g., diseases associated with expansions of trinucleotiderepeats such as polyglutamine (polyQ) repeat diseases, e.g.,Huntington's disease (HD), spinocerebellar ataxia (SCA1, SCA2, SCA3,SCA6, SCA7, and SCA17), spinal and bulbar muscular atrophy (SBMA),dentatorubropallidoluysian atrophy (DRPLA). An example of a neurologicaldisorder that does not appear to result in atrophy is DYT1 dystonia.

The RNAi molecules of the present invention can be generated by anymethod known to the art, for example, by in vitro transcription,recombinantly, or by synthetic means. In one example, the RNAi moleculescan be generated in vitro by using a recombinant enzyme, such as T7 RNApolymerase, and DNA oligonucleotide templates.

II. Nucleic Acid Molecules of the Invention

The terms “isolated and/or purified” refer to in vitro isolation of anucleic acid, e.g., a DNA or RNA molecule from its natural cellularenvironment, and from association with other components of the cell,such as nucleic acid or polypeptide, so that it can be sequenced,replicated, and/or expressed. For example, “isolated nucleic acid” maybe a DNA molecule containing less than 31 sequential nucleotides that istranscribed into an RNAi molecule. Such an isolated RNAi molecule may,for example, form a hairpin structure with a duplex 21 base pairs inlength that is complementary or hybridizes to a sequence in a gene ofinterest, and remains stably bound under stringent conditions (asdefined by methods well known in the art, e.g., in Sambrook and Russell,2001). Thus, the RNA or DNA is “isolated” in that it is free from atleast one contaminating nucleic acid with which it is normallyassociated in the natural source of the RNA or DNA and is preferablysubstantially free of any other mammalian RNA or DNA. The phrase “freefrom at least one contaminating source nucleic acid with which it isnormally associated” includes the case where the nucleic acid isreintroduced into the source or natural cell but is in a differentchromosomal location or is otherwise flanked by nucleic acid sequencesnot normally found in the source cell, e.g., in a vector or plasmid.

In addition to a DNA sequence encoding a siRNA, the nucleic acidmolecules of the invention include double-stranded interfering RNAmolecules, which are also useful to inhibit expression of a target gene.

As used herein, the term “recombinant nucleic acid”, e.g., “recombinantDNA sequence or segment” refers to a nucleic acid, e.g., to DNA, thathas been derived or isolated from any appropriate cellular source, thatmay be subsequently chemically altered in vitro, so that its sequence isnot naturally occurring, or corresponds to naturally occurring sequencesthat are not positioned as they would be positioned in a genome whichhas not been transformed with exogenous DNA. An example of preselectedDNA “derived” from a source would be a DNA sequence that is identifiedas a useful fragment within a given organism, and which is thenchemically synthesized in essentially pure form. An example of such DNA“isolated” from a source would be a useful DNA sequence that is excisedor removed from said source by chemical means, e.g., by the use ofrestriction endonucleases, so that it can be further manipulated, e.g.,amplified, for use in the invention, by the methodology of geneticengineering.

Thus, recovery or isolation of a given fragment of DNA from arestriction digest can employ separation of the digest on polyacrylamideor agarose gel by electrophoresis, identification of the fragment ofinterest by comparison of its mobility versus that of marker DNAfragments of known molecular weight, removal of the gel sectioncontaining the desired fragment, and separation of the gel from DNA.Therefore, “recombinant DNA” includes completely synthetic DNAsequences, semi-synthetic DNA sequences, DNA sequences isolated frombiological sources, and DNA sequences derived from RNA, as well asmixtures thereof.

Nucleic acid molecules having base substitutions (i.e., variants) areprepared by a variety of methods known in the art. These methodsinclude, but are not limited to, isolation from a natural source (in thecase of naturally occurring sequence variants) or preparation byoligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant version of the nucleic acid molecule.

Oligonucleotide-mediated mutagenesis is a method for preparingsubstitution variants. Briefly, nucleic acid encoding a siRNA can bealtered by hybridizing an oligonucleotide encoding the desired mutationto a DNA template, where the template is the single-stranded form of aplasmid or bacteriophage containing the unaltered or native genesequence. After hybridization, a DNA polymerase is used to synthesize anentire second complementary strand of the template that will thusincorporate the oligonucleotide primer, and will code for the selectedalteration in the nucleic acid encoding siRNA. Generally,oligonucleotides of at least 25 nucleotides in length are used. Anoptimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art.

The DNA template can be generated by those vectors that are eitherderived from bacteriophage M13 vectors (the commercially availableM13mp18 and M13mp19 vectors are suitable), or those vectors that containa single-stranded phage origin of replication. Thus, the DNA that is tobe mutated may be inserted into one of these vectors to generatesingle-stranded template. Production of the single-stranded template isdescribed in Chapter 3 of Sambrook and Russell, 2001. Alternatively,single-stranded DNA template may be generated by denaturingdouble-stranded plasmid (or other) DNA using standard techniques.

For alteration of the native DNA sequence (to generate amino acidsequence variants, for example), the oligonucleotide is hybridized tothe single-stranded template under suitable hybridization conditions. ADNA polymerizing enzyme, usually the Klenow fragment of DNA polymeraseI, is then added to synthesize the complementary strand of the templateusing the oligonucleotide as a primer for synthesis. A heteroduplexmolecule is thus formed such that one strand of DNA encodes the mutatedform of the DNA, and the other strand (the original template) encodesthe native, unaltered sequence of the DNA. This heteroduplex molecule isthen transformed into a suitable host cell, usually a prokaryote such asE. coli JM101. After the cells are grown, they are plated onto agaroseplates and screened using the oligonucleotide primer radiolabeled with32-phosphate to identify the bacterial colonies that contain the mutatedDNA. The mutated region is then removed and placed in an appropriatevector, generally an expression vector of the type typically employedfor transformation of an appropriate host.

The method described immediately above may be modified such that ahomoduplex molecule is created wherein both strands of the plasmidcontain the mutations(s). The modifications are as follows: Thesingle-stranded oligonucleotide is annealed to the single-strandedtemplate as described above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTTP), is combined with a modifiedthiodeoxyribocytosine called dCTP-(*S) (which can be obtained from theAmersham Corporation). This mixture is added to thetemplate-oligonucleotide complex. Upon addition of DNA polymerase tothis mixture, a strand of DNA identical to the template except for themutated bases is generated. In addition, this new strand of DNA willcontain dCTP-(*S) instead of dCTP, which serves to protect it fromrestriction endonuclease digestion.

After the template strand of the double-stranded heteroduplex is nickedwith an appropriate restriction enzyme, the template strand can bedigested with ExoIII nuclease or another appropriate nuclease past theregion that contains the site(s) to be mutagenized. The reaction is thenstopped to leave a molecule that is only partially single-stranded. Acomplete double-stranded DNA homoduplex is then formed using DNApolymerase in the presence of all four deoxyribonucleotidetriphosphates, ATP, and DNA ligase. This homoduplex molecule can then betransformed into a suitable host cell such as E. coli JM101.

There are well-established criteria for designing siRNAs. However, sincethe mechanism for siRNAs suppressing gene expression is not entirelyunderstood and siRNAs selected from different regions of the same genedo not work as equally effective, very often a number of siRNAs have tobe generated at the same time in order to compare their effectiveness.

III. Expression Cassettes of the Invention

To prepare expression cassettes, the recombinant DNA sequence or segmentmay be circular or linear, double-stranded or single-stranded.Generally, the DNA sequence or segment is in the form of chimeric DNA,such as plasmid DNA or a vector that can also contain coding regionsflanked by control sequences that promote the expression of therecombinant DNA present in the resultant transformed cell.

A “chimeric” vector or expression cassette, as used herein, means avector or cassette including nucleic acid sequences from at least twodifferent species, or has a nucleic acid sequence from the same speciesthat is linked or associated in a manner that does not occur in the“native” or wild type of the species.

Aside from recombinant DNA sequences that serve as transcription unitsfor an RNA transcript, or portions thereof, a portion of the recombinantDNA may be untranscribed, serving a regulatory or a structural function.For example, the recombinant DNA may have a promoter that is active inmammalian cells.

Other elements functional in the host cells, such as introns, enhancers,polyadenylation sequences and the like, may also be a part of therecombinant DNA. Such elements may or may not be necessary for thefunction of the DNA, but may provide improved expression of the DNA byaffecting transcription, stability of the siRNA, or the like. Suchelements may be included in the DNA as desired to obtain the optimalperformance of the siRNA in the cell.

Control sequences are DNA sequences necessary for the expression of anoperably linked coding sequence in a particular host organism. Thecontrol sequences that are suitable for prokaryotic cells, for example,include a promoter, and optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Operably linked nucleic acids are nucleic acids placed in a functionalrelationship with another nucleic acid sequence. For example, a promoteror enhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, operably linked DNA sequences are DNA sequencesthat are linked are contiguous. However, enhancers do not have to becontiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accord with conventionalpractice.

The recombinant DNA to be introduced into the cells may contain either aselectable marker gene or a reporter gene or both to facilitateidentification and selection of expressing cells from the population ofcells sought to be transfected or infected through viral vectors. Inother embodiments, the selectable marker may be carried on a separatepiece of DNA and used in a co-transfection procedure. Both selectablemarkers and reporter genes may be flanked with appropriate regulatorysequences to enable expression in the host cells. Useful selectablemarkers are known in the art and include, for example,antibiotic-resistance genes, such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Reportergenes that encode for easily assayable proteins are well known in theart. In general, a reporter gene is a gene that is not present in orexpressed by the recipient organism or tissue and that encodes a proteinwhose expression is manifested by some easily detectable property, e.g.,enzymatic activity. For example, reporter genes include thechloramphenicol acetyl transferase gene (cat) from Tn9 of E. coli andthe luciferase gene from firefly Photinus pyralis. Expression of thereporter gene is assayed at a suitable time after the DNA has beenintroduced into the recipient cells.

The general methods for constructing recombinant DNA that can transfecttarget cells are well known to those skilled in the art, and the samecompositions and methods of construction may be utilized to produce theDNA useful herein.

The recombinant DNA can be readily introduced into the host cells, e.g.,mammalian, bacterial, yeast or insect cells by transfection with anexpression vector composed of DNA encoding the siRNA by any procedureuseful for the introduction into a particular cell, e.g., physical orbiological methods, to yield a cell having the recombinant DNA stablyintegrated into its genome or existing as a episomal element, so thatthe DNA molecules, or sequences of the present invention are expressedby the host cell. Preferably, the DNA is introduced into host cells viaa vector. The host cell is preferably of eukaryotic origin, e.g., plant,mammalian, insect, yeast or fungal sources, but host cells ofnon-eukaryotic origin may also be employed.

Physical methods to introduce a preselected DNA into a host cell includecalcium phosphate precipitation, lipofection, particle bombardment,microinjection, electroporation, and the like. Biological methods tointroduce the DNA of interest into a host cell include the use of DNAand RNA viral vectors. For mammalian gene therapy, as described hereinbelow, it is desirable to use an efficient means of inserting a copygene into the host genome. Viral vectors, and especially retroviralvectors, have become the most widely used method for inserting genesinto mammalian, e.g., human cells. Other viral vectors can be derivedfrom poxviruses, herpes simplex virus I, adenoviruses andadeno-associated viruses, and the like.

As discussed above, a “transfected” or “transduced” host cell or cellline is one in which the genome has been altered or augmented by thepresence of at least one heterologous or recombinant nucleic acidsequence. The host cells of the present invention are typically producedby transfection with a DNA sequence in a plasmid expression vector, aviral expression vector, or as an isolated linear DNA sequence. Thetransfected DNA can become a chromosomally integrated recombinant DNAsequence, which is composed of sequence encoding the siRNA.

To confirm the presence of the recombinant DNA sequence in the hostcell, a variety of assays may be performed. Such assays include, forexample, “molecular biological” assays well known to those of skill inthe art, such as Southern and Northern blotting, RT-PCR and PCR;“biochemical” assays, such as detecting the presence or absence of aparticular peptide, e.g., by immunological means (ELISAs and Westernblots) or by assays described herein to identify agents falling withinthe scope of the invention.

To detect and quantitate RNA produced from introduced recombinant DNAsegments, RT-PCR may be employed. In this application of PCR, it isfirst necessary to reverse transcribe RNA into DNA, using enzymes suchas reverse transcriptase, and then through the use of conventional PCRtechniques amplify the DNA. In most instances PCR techniques, whileuseful, will not demonstrate integrity of the RNA product. Furtherinformation about the nature of the RNA product may be obtained byNorthern blotting. This technique demonstrates the presence of an RNAspecies and gives information about the integrity of that RNA. Thepresence or absence of an RNA species can also be determined using dotor slot blot Northern hybridizations. These techniques are modificationsof Northern blotting and only demonstrate the presence or absence of anRNA species.

While Southern blotting and PCR may be used to detect the recombinantDNA segment in question, they do not provide information as to whetherthe preselected DNA segment is being expressed. Expression may beevaluated by specifically identifying the peptide products of theintroduced recombinant DNA sequences or evaluating the phenotypicchanges brought about by the expression of the introduced recombinantDNA segment in the host cell.

The instant invention provides a cell expression system for expressingexogenous nucleic acid material in a mammalian recipient. The expressionsystem, also referred to as a “genetically modified cell,” comprises acell and an expression vector for expressing the exogenous nucleic acidmaterial. The genetically modified cells are suitable for administrationto a mammalian recipient, where they replace the endogenous cells of therecipient. Thus, the preferred genetically modified cells arenon-immortalized and are non-tumorigenic.

According to one embodiment, the cells are transfected or otherwisegenetically modified ex vivo. The cells are isolated from a mammal(preferably a human), nucleic acid introduced (i.e., transduced ortransfected in vitro) with a vector for expressing a heterologous (e.g.,recombinant) gene encoding the therapeutic agent, and then administeredto a mammalian recipient for delivery of the therapeutic agent in situ.The mammalian recipient may be a human and the cells to be modified areautologous cells, i.e., the cells are isolated from the mammalianrecipient.

According to another embodiment, the cells are transfected or transducedor otherwise genetically modified in vivo. The cells from the mammalianrecipient are transduced or transfected in vivo with a vector containingexogenous nucleic acid material for expressing a heterologous (e.g.,recombinant) gene encoding a therapeutic agent and the therapeutic agentis delivered in situ.

As used herein, “exogenous nucleic acid material” refers to a nucleicacid or an oligonucleotide, either natural or synthetic, which is notnaturally found in the cells; or if it is naturally found in the cells,is modified from its original or native form. Thus, “exogenous nucleicacid material” includes, for example, a non-naturally occurring nucleicacid that can be transcribed into an anti-sense RNA, a siRNA, as well asa “heterologous gene” (i.e., a gene encoding a protein that is notexpressed or is expressed at biologically insignificant levels in anaturally-occurring cell of the same type). To illustrate, a syntheticor natural gene encoding human erythropoietin (EPO) would be considered“exogenous nucleic acid material” with respect to human peritonealmesothelial cells since the latter cells do not naturally express EPO.Still another example of “exogenous nucleic acid material” is theintroduction of only part of a gene to create a recombinant gene, suchas combining an regulatable promoter with an endogenous coding sequencevia homologous recombination.

IV. MicroRNA Shuttles for RNAi

miRNAs are small cellular RNAs (˜22 nt) that are processed fromprecursor stem loop transcripts. Known miRNA stem loops can be modifiedto contain RNAi sequences specific for genes of interest. miRNAmolecules can be preferable over shRNA molecules because miRNAs areendogenously expressed. Therefore, miRNA molecules are unlikely toinduce dsRNA-responsive interferon pathways, they are processed moreefficiently than shRNAs, and they have been shown to silence 80% moreeffectively.

Also, the promoter roles are different for miRNA molecules as comparedto shRNA molecules. Tissue-specific, inducible expression of shRNAsinvolves truncation of polII promoters to the transcription start site.In contrast, miRNAs can be expressed from any polII promoter because thetranscription start and stop sites can be relatively arbitrary.

V. Methods for Introducing the Expression Cassettes of the Inventioninto Cells

The condition amenable to gene inhibition therapy may be a prophylacticprocess, i.e., a process for preventing disease or an undesired medicalcondition. Thus, the instant invention embraces a system for deliveringsiRNA that has a prophylactic function (i.e., a prophylactic agent) tothe mammalian recipient.

The inhibitory nucleic acid material (e.g., an expression cassetteencoding siRNA directed to a gene of interest) can be introduced intothe cell ex vivo or in vivo by genetic transfer methods, such astransfection or transduction, to provide a genetically modified cell.Various expression vectors (i.e., vehicles for facilitating delivery ofexogenous nucleic acid into a target cell) are known to one of ordinaryskill in the art.

As used herein, “transfection of cells” refers to the acquisition by acell of new nucleic acid material by incorporation of added DNA. Thus,transfection refers to the insertion of nucleic acid into a cell usingphysical or chemical methods. Several transfection techniques are knownto those of ordinary skill in the art including calcium phosphate DNAco-precipitation, DEAE-dextran, electroporation, cationicliposome-mediated transfection, tungsten particle-facilitatedmicroparticle bombardment, and strontium phosphate DNA co-precipitation.

In contrast, “transduction of cells” refers to the process oftransferring nucleic acid into a cell using a DNA or RNA virus. A RNAvirus (i.e., a retrovirus) for transferring a nucleic acid into a cellis referred to herein as a transducing chimeric retrovirus. Exogenousnucleic acid material contained within the retrovirus is incorporatedinto the genome of the transduced cell. A cell that has been transducedwith a chimeric DNA virus (e.g., an adenovirus carrying a cDNA encodinga therapeutic agent), will not have the exogenous nucleic acid materialincorporated into its genome but will be capable of expressing theexogenous nucleic acid material that is retained extrachromosomallywithin the cell.

The exogenous nucleic acid material can include the nucleic acidencoding the siRNA together with a promoter to control transcription.The promoter characteristically has a specific nucleotide sequencenecessary to initiate transcription. The exogenous nucleic acid materialmay further include additional sequences (i.e., enhancers) required toobtain the desired gene transcription activity. For the purpose of thisdiscussion an “enhancer” is simply any non-translated DNA sequence thatworks with the coding sequence (in cis) to change the basaltranscription level dictated by the promoter. The exogenous nucleic acidmaterial may be introduced into the cell genome immediately downstreamfrom the promoter so that the promoter and coding sequence areoperatively linked so as to permit transcription of the coding sequence.An expression vector can include an exogenous promoter element tocontrol transcription of the inserted exogenous gene. Such exogenouspromoters include both constitutive and regulatable promoters.

Naturally-occurring constitutive promoters control the expression ofessential cell functions. As a result, a nucleic acid sequence under thecontrol of a constitutive promoter is expressed under all conditions ofcell growth. Constitutive promoters include the promoters for thefollowing genes which encode certain constitutive or “housekeeping”functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolatereductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK),pyruvate kinase, phosphoglycerol mutase, the beta□-actin promoter, andother constitutive promoters known to those of skill in the art. Inaddition, many viral promoters function constitutively in eukaryoticcells. These include: the early and late promoters of SV40; the longterminal repeats (LTRs) of Moloney Leukemia Virus and otherretroviruses; and the thymidine kinase promoter of Herpes Simplex Virus,among many others.

Nucleic acid sequences that are under the control of regulatablepromoters are expressed only or to a greater or lesser degree in thepresence of an inducing or repressing agent, (e.g., transcription undercontrol of the metallothionein promoter is greatly increased in presenceof certain metal ions). Regulatable promoters include responsiveelements (REs) that stimulate transcription when their inducing factorsare bound. For example, there are REs for serum factors, steroidhormones, retinoic acid, cyclic AMP, and tetracycline and doxycycline.Promoters containing a particular RE can be chosen in order to obtain anregulatable response and in some cases, the RE itself may be attached toa different promoter, thereby conferring regulatability to the encodednucleic acid sequence. Thus, by selecting the appropriate promoter(constitutive versus regulatable; strong versus weak), it is possible tocontrol both the existence and level of expression of a nucleic acidsequence in the genetically modified cell. If the nucleic acid sequenceis under the control of an regulatable promoter, delivery of thetherapeutic agent in situ is triggered by exposing the geneticallymodified cell in situ to conditions for permitting transcription of thenucleic acid sequence, e.g., by intraperitoneal injection of specificinducers of the regulatable promoters which control transcription of theagent. For example, in situ expression of a nucleic acid sequence underthe control of the metallothionein promoter in genetically modifiedcells is enhanced by contacting the genetically modified cells with asolution containing the appropriate (i.e., inducing) metal ions in situ.

Accordingly, the amount of siRNA generated in situ is regulated bycontrolling such factors as the nature of the promoter used to directtranscription of the nucleic acid sequence, (i.e., whether the promoteris constitutive or regulatable, strong or weak) and the number of copiesof the exogenous nucleic acid sequence encoding a siRNA sequence thatare in the cell.

In one embodiment of the present invention, an expression cassette maycontain a pol II promoter that is operably linked to a nucleic acidsequence encoding a siRNA. Thus, the pol II promoter, i.e., a RNApolymerase II dependent promoter, initiates the transcription of thesiRNA. In another embodiment, the pol II promoter is regulatable.

A pol II promoter may be used in its entirety, or a portion or fragmentof the promoter sequence may be used in which the portion maintains thepromoter activity. As discussed herein, pol II promoters are known to askilled person in the art and include the promoter of anyprotein-encoding gene, e.g., an endogenously regulated gene or aconstitutively expressed gene. For example, the promoters of genesregulated by cellular physiological events, e.g., heat shock, oxygenlevels and/or carbon monoxide levels, e.g., in hypoxia, may be used inthe expression cassettes of the invention. In addition, the promoter ofany gene regulated by the presence of a pharmacological agent, e.g.,tetracycline and derivatives thereof, as well as heavy metal ions andhormones may be employed in the expression cassettes of the invention.In an embodiment of the invention, the pol II promoter can be the CMVpromoter or the RSV promoter. In another embodiment, the pol II promoteris the CMV promoter.

As discussed above, a pol II promoter of the invention may be onenaturally associated with an endogenously regulated gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. The pol II promoter of theexpression cassette can be, for example, the same pol II promoterdriving expression of the targeted gene of interest. Alternatively, thenucleic acid sequence encoding the RNAi molecule may be placed under thecontrol of a recombinant or heterologous pol II promoter, which refersto a promoter that is not normally associated with the targeted gene'snatural environment. Such promoters include promoters isolated from anyeukaryotic cell, and promoters not “naturally occurring,” i.e.,containing different elements of different transcriptional regulatoryregions, and/or mutations that alter expression. In addition toproducing nucleic acid sequences of promoters synthetically, sequencesmay be produced using recombinant cloning and/or nucleic acidamplification technology, including PCR, in connection with thecompositions disclosed herein.

In one embodiment, a pol II promoter that effectively directs theexpression of the siRNA in the cell type, organelle, and organism chosenfor expression will be employed. Those of ordinary skill in the art ofmolecular biology generally know the use of promoters for proteinexpression. The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The identity of tissue-specific promoters, as well as assays tocharacterize their activity, is well known to those of ordinary skill inthe art.

In addition to at least one promoter and at least one heterologousnucleic acid sequence encoding the siRNA, the expression vector mayinclude a selection gene, for example, a neomycin resistance gene, forfacilitating selection of cells that have been transfected or transducedwith the expression vector.

Cells can also be transfected with two or more expression vectors, atleast one vector containing the nucleic acid sequence(s) encoding thesiRNA(s), the other vector containing a selection gene. The selection ofa suitable promoter, enhancer, selection gene and/or signal sequence isdeemed to be within the scope of one of ordinary skill in the artwithout undue experimentation.

The following discussion is directed to various utilities of the instantinvention. For example, the instant invention has utility as anexpression system suitable for silencing the expression of gene(s) ofinterest.

The instant invention also provides methods for genetically modifyingcells of a mammalian recipient in vivo. According to one embodiment, themethod comprises introducing an expression vector for expressing a siRNAsequence in cells of the mammalian recipient in situ by, for example,injecting the vector into the recipient.

VI. Delivery Vehicles for the Expression Cassettes of the Invention

Delivery of compounds into tissues and across the blood-brain barriercan be limited by the size and biochemical properties of the compounds.Currently, efficient delivery of compounds into cells in vivo can beachieved only when the molecules are small (usually less than 600Daltons). Gene transfer for the correction of inborn errors ofmetabolism and neurodegenerative diseases of the central nervous system(CNS), and for the treatment of cancer has been accomplished withrecombinant adenoviral vectors.

The selection and optimization of a particular expression vector forexpressing a specific siRNA in a cell can be accomplished by obtainingthe nucleic acid sequence of the siRNA, possibly with one or moreappropriate control regions (e.g., promoter, insertion sequence);preparing a vector construct comprising the vector into which isinserted the nucleic acid sequence encoding the siRNA; transfecting ortransducing cultured cells in vitro with the vector construct; anddetermining whether the siRNA is present in the cultured cells.

Vectors for cell gene therapy include viruses, such asreplication-deficient viruses (described in detail below). Exemplaryviral vectors are derived from Harvey Sarcoma virus, ROUS Sarcoma virus,(MPSV), Moloney murine leukemia virus and DNA viruses (e.g.,adenovirus).

Replication-deficient retroviruses are capable of directing synthesis ofall virion proteins, but are incapable of making infectious particles.Accordingly, these genetically altered retroviral expression vectorshave general utility for high-efficiency transduction of nucleic acidsequences in cultured cells, and specific utility for use in the methodof the present invention. Such retroviruses further have utility for theefficient transduction of nucleic acid sequences into cells in vivo.Retroviruses have been used extensively for transferring nucleic acidmaterial into cells. Protocols for producing replication-deficientretroviruses (including the steps of incorporation of exogenous nucleicacid material into a plasmid, transfection of a packaging cell line withplasmid, production of recombinant retroviruses by the packaging cellline, collection of viral particles from tissue culture media, andinfection of the target cells with the viral particles) are well knownin the art.

An advantage of using retroviruses for gene therapy is that the virusesinsert the nucleic acid sequence encoding the siRNA into the host cellgenome, thereby permitting the nucleic acid sequence encoding the siRNAto be passed on to the progeny of the cell when it divides. Promotersequences in the LTR region have can enhance expression of an insertedcoding sequence in a variety of cell types. Some disadvantages of usinga retrovirus expression vector are (1) insertional mutagenesis, i.e.,the insertion of the nucleic acid sequence encoding the siRNA into anundesirable position in the target cell genome which, for example, leadsto unregulated cell growth and (2) the need for target cellproliferation in order for the nucleic acid sequence encoding the siRNAcarried by the vector to be integrated into the target genome.

Another viral candidate useful as an expression vector fortransformation of cells is the adenovirus, a double-stranded DNA virus.The adenovirus is infective in a wide range of cell types, including,for example, muscle and endothelial cells.

Adenoviruses (Ad) are double-stranded linear DNA viruses with a 36 kbgenome. Several features of adenovirus have made them useful astransgene delivery vehicles for therapeutic applications, such asfacilitating in vivo gene delivery. Recombinant adenovirus vectors havebeen shown to be capable of efficient in situ gene transfer toparenchymal cells of various organs, including the lung, brain,pancreas, gallbladder, and liver. This has allowed the use of thesevectors in methods for treating inherited genetic diseases, such ascystic fibrosis, where vectors may be delivered to a target organ. Inaddition, the ability of the adenovirus vector to accomplish in situtumor transduction has allowed the development of a variety ofanticancer gene therapy methods for non-disseminated disease. In thesemethods, vector containment favors tumor cell-specific transduction.

Like the retrovirus, the adenovirus genome is adaptable for use as anexpression vector for gene therapy, i.e., by removing the geneticinformation that controls production of the virus itself. Because theadenovirus functions in an extrachromosomal fashion, the recombinantadenovirus does not have the theoretical problem of insertionalmutagenesis.

Several approaches traditionally have been used to generate therecombinant adenoviruses. One approach involves direct ligation ofrestriction endonuclease fragments containing a nucleic acid sequence ofinterest to portions of the adenoviral genome. Alternatively, thenucleic acid sequence of interest may be inserted into a defectiveadenovirus by homologous recombination results. The desired recombinantsare identified by screening individual plaques generated in a lawn ofcomplementation cells.

Most adenovirus vectors are based on the adenovirus type 5 (Ad5)backbone in which an expression cassette containing the nucleic acidsequence of interest has been introduced in place of the early region 1(E1) or early region 3 (E3). Viruses in which E1 has been deleted aredefective for replication and are propagated in human complementationcells (e.g., 293 or 911 cells), which supply the missing gene E1 and pIXin trans.

In one embodiment of the present invention, one will desire to generatean RNAi molecule in a brain cell or brain tissue. A suitable vector forthis application is an FIV vector or an AAV vector. For example, one mayuse AAV5. Also, one may apply poliovirus or HSV vectors.

Application of siRNA is generally accomplished by transfection ofsynthetic siRNAs, in vitro synthesized RNAs, or plasmids expressingshort hairpin RNAs (shRNAs). More recently, viruses have been employedfor in vitro studies and to generate transgenic mouse knock-downs oftargeted genes. Recombinant adenovirus, adeno-associated virus (AAV) andfeline immunodeficiency virus (FIV) can be used to deliver genes invitro and in vivo. Each has its own advantages and disadvantages.Adenoviruses are double stranded DNA viruses with large genomes (36 kb)and have been engineered to accommodate expression cassettes in distinctregions. The inventors previously have used recombinant adenovirusesexpressing siRNAs to demonstrate successful viral-mediated genesuppression in brain.

Adeno-associated viruses have encapsidated genomes, similar to Ad, butare smaller in size and packaging capacity (˜30 nm vs. ˜100 nm;packaging limit of ˜4.5 kb). AAV contain single stranded DNA genomes ofthe + or the − strand. Eight serotypes of AAV (1-8) have been studiedextensively, three of which have been evaluated in the brain. Animportant consideration for the present application is that AAV5transduces striatal and cortical neurons, and is not associated with anyknown pathologies.

Adeno associated virus (AAV) is a small nonpathogenic virus of theparvoviridae family. AAV is distinct from the other members of thisfamily by its dependence upon a helper virus for replication. In theabsence of a helper virus, AAV may integrate in a locus specific mannerinto the q arm of chromosome 19. The approximately 5 kb genome of AAVconsists of one segment of single stranded DNA of either plus or minuspolarity. The ends of the genome are short inverted terminal repeatswhich can fold into hairpin structures and serve as the origin of viralDNA replication. Physically, the parvovirus virion is non-enveloped andits icosohedral capsid is approximately 20 nm in diameter.

To-date seven serologically distinct AAVs have been identified and fivehave been isolated from humans or primates and are referred to as AAVtypes 1-5. The most extensively studied of these isolates is AAV type 2(AAV2). The genome of AAV2 is 4680 nucleotides in length and containstwo open reading frames (ORFs). The left ORF encodes the non-structuralRep proteins, Rep40, Rep 52, Rep68 and Rep 78, which are involved inregulation of replication and transcription in addition to theproduction of single-stranded progeny genomes. Furthermore, two of theRep proteins have been associated with the possible integration of AAVgenomes into a region of the q-arm of human chromosome 19. Rep68/78 hasalso been shown to possess NTP binding activity as well as DNA and RNAhelicase activities. The Rep proteins possess a nuclear localizationsignal as well as several potential phosphorylation sites. Mutation ofone of these kinase sites resulted in a loss of replication activity.

The ends of the genome are short inverted terminal repeats which havethe potential to fold into T-shaped hairpin structures that serve as theorigin of viral DNA replication. Within the ITR region two elements havebeen described which are central to the function of the ITR, a GAGCrepeat motif and the terminal resolution site (trs). The repeat motifhas been shown to bind Rep when the ITR is in either a linear or hairpinconformation. This binding serves to position Rep68/78 for cleavage atthe trs which occurs in a site- and strand-specific manner. In additionto their role in replication, these two elements appear to be central toviral integration. Contained within the chromosome 19 integration locusis a Rep binding site with an adjacent trs. These elements have beenshown to be functional and necessary for locus specific integration.

The AAV2 virion is a non-enveloped, icosohedral particle approximately25 nm in diameter, consisting of three related proteins referred to asVPI,2 and 3. The right ORF encodes the capsid proteins, VP1, VP2, andVP3. These proteins are found in a ratio of 1:1:10 respectively and areall derived from the right-hand ORF. The capsid proteins differ fromeach other by the use of alternative splicing and an unusual startcodon. Deletion analysis has shown that removal or alteration of VP1which is translated from an alternatively spliced message results in areduced yield of infections particles. Mutations within the VP3 codingregion result in the failure to produce any single-stranded progeny DNAor infectious particles.

The following features of AAV have made it an attractive vector for genetransfer. AAV vectors have been shown in vitro to stably integrate intothe cellular genome; possess a broad host range; transduce both dividingand non dividing cells in vitro and in vivo and maintain high levels ofexpression of the transduced genes. Viral particles are heat stable,resistant to solvents, detergents, changes in pH, temperature, and canbe concentrated on CsCl gradients. Integration of AAV provirus is notassociated with any long term negative effects on cell growth ordifferentiation. The ITRs have been shown to be the only cis elementsrequired for replication, packaging and integration and may contain somepromoter activities.

Further provided by this invention are chimeric viruses where AAV can becombined with herpes virus, herpes virus amplicons, baculovirus or otherviruses to achieve a desired tropism associated with another virus. Forexample, the AAV4 ITRs could be inserted in the herpes virus and cellscould be infected. Post-infection, the ITRs of AAV4 could be acted on byAAV4 rep provided in the system or in a separate vehicle to rescue AAV4from the genome. Therefore, the cellular tropism of the herpes simplexvirus can be combined with AAV4 rep mediated targeted integration. Otherviruses that could be utilized to construct chimeric viruses includelentivirus, retrovirus, pseudotyped retroviral vectors, and adenoviralvectors.

Also provided by this invention are variant AAV vectors. For example,the sequence of a native AAV, such as AAV5, can be modified atindividual nucleotides. The present invention includes native and mutantAAV vectors. The present invention further includes all AAV serotypes.

FIV is an enveloped virus with a strong safety profile in humans;individuals bitten or scratched by FIV-infected cats do not seroconvertand have not been reported to show any signs of disease. Like AAV, FIVprovides lasting transgene expression in mouse and nonhuman primateneurons, and transduction can be directed to different cell types bypseudotyping, the process of exchanging the virus' native envelope foran envelope from another virus.

Thus, as will be apparent to one of ordinary skill in the art, a varietyof suitable viral expression vectors are available for transferringexogenous nucleic acid material into cells. The selection of anappropriate expression vector to express a therapeutic agent for aparticular condition amenable to gene silencing therapy and theoptimization of the conditions for insertion of the selected expressionvector into the cell, are within the scope of one of ordinary skill inthe art without the need for undue experimentation.

In another embodiment, the expression vector is in the form of aplasmid, which is transferred into the target cells by one of a varietyof methods: physical (e.g., microinjection, electroporation, scrapeloading, microparticle bombardment) or by cellular uptake as a chemicalcomplex (e.g., calcium or strontium co-precipitation, complexation withlipid, complexation with ligand). Several commercial products areavailable for cationic liposome complexation including Lipofectin™(Gibco-BRL, Gaithersburg, Md.) and Transfectam™ (ProMega, Madison,Wis.). However, the efficiency of transfection by these methods ishighly dependent on the nature of the target cell and accordingly, theconditions for optimal transfection of nucleic acids into cells usingthe above-mentioned procedures must be optimized. Such optimization iswithin the scope of one of ordinary skill in the art without the needfor undue experimentation.

VII. Diseases and Conditions Amendable to the Methods of the Invention

In the certain embodiments of the present invention, a mammalianrecipient to an expression cassette of the invention has a conditionthat is amenable to gene silencing therapy. As used herein, “genesilencing therapy” refers to administration to the recipient exogenousnucleic acid material encoding a therapeutic siRNA and subsequentexpression of the administered nucleic acid material in situ. Thus, thephrase “condition amenable to siRNA therapy” embraces conditions such asgenetic diseases (i.e., a disease condition that is attributable to oneor more gene defects), acquired pathologies (i.e., a pathologicalcondition that is not attributable to an inborn defect), cancers,neurodegenerative diseases, e.g., trinucleotide repeat disorders, andprophylactic processes (i.e., prevention of a disease or of an undesiredmedical condition). A gene “associated with a condition” is a gene thatis either the cause, or is part of the cause, of the condition to betreated. Examples of such genes include genes associated with aneurodegenerative disease (e.g., a trinucleotide-repeat disease such asa disease associated with polyglutamine repeats, Huntington's disease,and several spinocerebellar ataxias), and genes encoding ligands forchemokines involved in the migration of a cancer cells, or chemokinereceptor. Also siRNA expressed from viral vectors may be used for invivo antiviral therapy using the vector systems described.

Accordingly, as used herein, the term “therapeutic siRNA” refers to anysiRNA that has a beneficial effect on the recipient. Thus, “therapeuticsiRNA” embraces both therapeutic and prophylactic siRNA.

Differences between alleles that are amenable to targeting by siRNAinclude disease-causing mutations as well as polymorphisms that are notthemselves mutations, but may be linked to a mutation or associated witha predisposition to a disease state. An example of a targetablepolymorphism that is not itself a mutation is the polymorphism in exon58 associated with Huntington's disease.

Single nucleotide polymorphisms comprise most of the genetic diversitybetween humans. The major risk factor for developing Alzheimer's diseaseis the presence of a particular polymorphism in the apolipoprotein Egene.

Single nucleotide polymorphisms comprise most of the genetic diversitybetween humans, and that many disease genes, including the HD gene inHuntington's disease, contain numerous single nucleotide or multiplenucleotide polymorphisms that could be separately targeted in one allelevs. the other. The major risk factor for developing Alzheimer's diseaseis the presence of a particular polymorphism in the apolipoprotein Egene.

A. Gene Defects

A number of diseases caused by gene defects have been identified. Forexample, this strategy can be applied to a major class of disablingneurological disorders. For example this strategy can be applied to thepolyglutamine diseases, as is demonstrated by the reduction ofpolyglutamine aggregation in cells following application of thestrategy. The neurodegenerative disease may be a trinucleotide-repeatdisease, such as a disease associated with polyglutamine repeats,including Huntington's disease, and several spinocerebellar ataxias.Additionally, this strategy can be applied to a non-degenerativeneurological disorder, such as DYT1 dystonia.

B. Acquired Pathologies

As used herein, “acquired pathology” refers to a disease or syndromemanifested by an abnormal physiological, biochemical, cellular,structural, or molecular biological state. For example, the diseasecould be a viral disease, such as hepatitis or AIDS.

C. Cancers

The condition amenable to gene silencing therapy alternatively can be agenetic disorder or an acquired pathology that is manifested by abnormalcell proliferation, e.g., cancer. According to this embodiment, theinstant invention is useful for silencing a gene involved in neoplasticactivity. The present invention can also be used to inhibitoverexpression of one or several genes. The present invention can beused to treat neuroblastoma, medulloblastoma, or glioblastoma.

IX. Dosages, Formulations and Routes of Administration of the Agents ofthe Invention

The agents of the invention are preferably administered so as to resultin a reduction in at least one symptom associated with a disease. Theamount administered will vary depending on various factors including,but not limited to, the composition chosen, the particular disease, theweight, the physical condition, and the age of the mammal, and whetherprevention or treatment is to be achieved. Such factors can be readilydetermined by the clinician employing animal models or other testsystems, which are well known to the art.

Administration of siRNA may be accomplished through the administrationof the nucleic acid molecule encoding the RNAi molecule. Pharmaceuticalformulations, dosages and routes of administration for nucleic acids aregenerally known in the art.

The present invention envisions treating a disease, for example, aneurodegenerative disease, in a mammal by the administration of anagent, e.g., a nucleic acid composition, an expression vector, or aviral particle of the invention. Administration of the therapeuticagents in accordance with the present invention may be continuous orintermittent, depending, for example, upon the recipient's physiologicalcondition, whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of the agents of the invention may be essentiallycontinuous over a preselected period of time or may be in a series ofspaced doses. Both local and systemic administration is contemplated.

One or more suitable unit dosage forms having the therapeutic agent(s)of the invention, which, as discussed below, may optionally beformulated for sustained release (for example using microencapsulation),can be administered by a variety of routes including parenteral,including by intravenous and intramuscular routes, as well as by directinjection into the diseased tissue. For example, the therapeutic agentmay be directly injected into the brain. Alternatively the therapeuticagent may be introduced intrathecally for brain and spinal cordconditions. In another example, the therapeutic agent may be introducedintramuscularly for viruses that traffic back to affected neurons frommuscle, such as AAV, lentivirus and adenovirus. The formulations may,where appropriate, be conveniently presented in discrete unit dosageforms and may be prepared by any of the methods well known to pharmacy.Such methods may include the step of bringing into association thetherapeutic agent with liquid carriers, solid matrices, semi-solidcarriers, finely divided solid carriers or combinations thereof, andthen, if necessary, introducing or shaping the product into the desireddelivery system.

When the therapeutic agents of the invention are prepared foradministration, they are preferably combined with a pharmaceuticallyacceptable carrier, diluent or excipient to form a pharmaceuticalformulation, or unit dosage form. The total active ingredients in suchformulations include from 0.1 to 99.9% by weight of the formulation. A“pharmaceutically acceptable” is a carrier, diluent, excipient, and/orsalt that is compatible with the other ingredients of the formulation,and not deleterious to the recipient thereof. The active ingredient foradministration may be present as a powder or as granules, as a solution,a suspension or an emulsion.

Pharmaceutical formulations containing the therapeutic agents of theinvention can be prepared by procedures known in the art using wellknown and readily available ingredients. The therapeutic agents of theinvention can also be formulated as solutions appropriate for parenteraladministration, for instance by intramuscular, subcutaneous orintravenous routes.

The pharmaceutical formulations of the therapeutic agents of theinvention can also take the form of an aqueous or anhydrous solution ordispersion, or alternatively the form of an emulsion or suspension.

Thus, the therapeutic agent may be formulated for parenteraladministration (e.g., by injection, for example, bolus injection orcontinuous infusion) and may be presented in unit dose form in ampules,pre-filled syringes, small volume infusion containers or in multi-dosecontainers with an added preservative. The active ingredients may takesuch forms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredients may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilization from solution, for constitution witha suitable vehicle, e.g., sterile, pyrogen-free water, before use.

It will be appreciated that the unit content of active ingredient oringredients contained in an individual aerosol dose of each dosage formneed not in itself constitute an effective amount for treating theparticular indication or disease since the necessary effective amountcan be reached by administration of a plurality of dosage units.Moreover, the effective amount may be achieved using less than the dosein the dosage form, either individually, or in a series ofadministrations.

The pharmaceutical formulations of the present invention may include, asoptional ingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, and salts of the type that arewell-known in the art. Specific non-limiting examples of the carriersand/or diluents that are useful in the pharmaceutical formulations ofthe present invention include water and physiologically acceptablebuffered saline solutions such as phosphate buffered saline solutions pH7.0-8.0. saline solutions and water.

The invention will now be illustrated by the following non-limitingExamples.

Example 1 RNAi-Mediated Silencing of Genes

The inventors have previously shown that genes can be silenced in anallele-specific manner. They have also demonstrated that viral-mediateddelivery of siRNA can specifically reduce expression of targeted genesin various cell types, both in vitro and in vivo. This strategy was thenapplied to reduce expression of a neurotoxic polyglutamine diseaseprotein. The ability of viral vectors to transduce cells efficiently invivo, coupled with the efficacy of virally expressed siRNA shown here,extends the application of siRNA to viral-based therapies and in vivotargeting experiments that aim to define the function of specific genes.

Huntington's disease (HD) is one of several dominant neurodegenerativediseases that result from a similar toxic gain of function mutation inthe disease protein: expansion of a polyglutamine (polyQ)-encodingtract. It is well established that for HD and other polyglutaminediseases, the length of the expansion correlates inversely with age ofdisease onset. Animal models for HD have provided important clues as tohow mutant huntingtin (htt) induces pathogenesis. Currently, noneuroprotective treatment exists for HD. RNA interference has emerged asa leading candidate approach to reduce expression of disease genes bytargeting the encoding mRNA for degradation.

Although the effect of partial reduction of wildtype htt in adultneurons is unknown, it is advantageous to target only mutant htt fordegradation, if possible. Disease allele-specific RNAi are designedusing approaches that led to allele specific silencing for otherneurogenetic disease models. This allows directed silencing of themutant, disease-causing expanded allele, leaving the normal alleleintact.

Constitutive expression of shRNA can prevent the neuropathological andbehavioral phenotypes in a mouse model of Spinocerebellar Ataxia type I,a related polyQ disease. However, the constitutive expression of shRNAmay not be necessary, particularly for pathologies that take many yearsto develop but may be cleared in a few weeks or months. For this reason,and to reduce long-term effects that may arise if nonspecific silencingor activation of interferon responses is noted, controlled expressionmay be very important. In order to regulate RNAi for diseaseapplication, doxycycline-responsive vectors have been developed forcontrolled silencing in vitro.

Most eukaryotes encode a substantial number of small noncoding RNAstermed micro RNAs (miRNAs). mir-30 is a 22-nucleotide human miRNA thatcan be naturally processed from a longer transcript bearing the proposedmiR-30 stem-loop precursor. mir-30 can translationally inhibit anmRNA-bearing artificial target sites. The mir-30 precursor stem can besubstituted with a heterologous stem, which can be processed to yieldnovel miRNAs and can block the expression of endogenous mRNAs.

Two strategies are possible to target a particular sequence, such as thegene involved in Huntington's Disease (FIGS. 1A and 1B). One can developnon-allele specific RNAi molecules, and candidates based on 8.2inhibitory RNAs have been developed. Alternatively, one can developallele-specific RNAi molecules. The inventors have worked to developRNAi molecules that target several key single nucleotide polymorphisms(SNPs). These RNAi molecules, however, may be limited to the treatmentof specific families/patients.

Another approach, which is the approach used in the present invention,the inventors targeted the expansion region. This approach has theadvantage of being able to treat entire HD populations, and not justthose with specific SNPs. These RNAi molecules are different becauseinstead of targeting a SNP for allele specificity, these sequences takeadvantage of structural integrity at the sites flanking the expansionregion. The siRNA data shows that they are effective. The presentinventors have also moved them into miRNA expression vectors, which werealso effective.

The inventors have generated and tested the following RNAi molecules:

siRNA Sequence HDAS 07 AUGAAGGCCUUCGAGUCCCUC (SEQ ID NO: 1) HDAS 18GGCGACCCUGGAAAAGCUGAU (SEQ ID NO: 2) HDAS 19UGGCGACCCUGGAAAAGCUGA (SEQ ID NO: 3) HDAS 20AUGGCGACCCUGGAAAAGCUG (SEQ ID NO: 4) Sequence miHD7A1 (SEQ ID NO: 5)AAAACUCGAGUGAGCGCUGAAGGCCUUCGAGUCCCUCA

UGAGGGACUCGAAGGCCUUCAUCGCCUACUAGUAAAA Sequence miHD7A2 (SEQ ID NO: 6)AAAACUCGAGUGAGCGCUGAAGGCCUUCGAGUCUUUUA

UGAGGGACUCGAAGGCCUUCAUCGCCUACUAGUAAAA Sequence miHD7B1 (SEQ ID NO: 7)AAAACUCGAGUGAGCGCAUGAAGGCCUUCGAGUCCCUC

GAGGGACUCGAAGGCCUUCAUCCGCCUACUAGUAAAA Sequence miHD7B2 (SEQ ID NO: 8)AAAACUCGAGUGAGCGCAUGAAGGCCUUCGAGUCUUUU

GAGGGACUCGAAGGCCUUCAUCCGCCUACUAGUAAAA

The different fonts show the various parts of the miRNA. In sequentialorder, the stem sequence of the miRNA is shown in bold, then the sensestrand in regular type, then the loop sequence in bold italics, then theanti-sense strand in regular type, and last, part of stem sequence inbold.

The inventors generated constructs to assess allele-specific silencingof Htt (FIGS. 2A and 2B). Two plasmids were generated expressingfull-length wild type (FIG. 2A, pCMV-FLHtt 18Q-Flag) or mutanthuntingtin (FIG. 2B, pCMV-FLHtt 83Q-V5). Wild type and mutantfull-length huntingtin are expressed under the control of the CMVpromoter and each cDNA have distinct epitope tags to differentiate itsexpression by western blot. To normalize transfection efficiencieseither renilla (WT htt) or firefly (mutant htt) luciferase were includedon the same plasmid. This design allowed assessment of allelespecificity in the same cell after co-transfection.

Western blot and Q-PCR results indicate that the candidate siRNAs wereallele-specific in targeting mutant Htt, but not wild type Htt (FIGS.3A-3C). HEK293 cells were co-transfected with plasmids expressing wildtype and mutant huntingtin and with different siRNA sequence. Total RNAand protein lysates were obtained 24 hours after transfection. Afterscreening by Q-PCR and western blot, some of the siRNA design sequenceswere observed to preferentially silence the mutant allele. FIG. 3A showswild type Htt and FIG. 3B shows mutant Htt. As seen in FIG. 3C, siRNAsequence number 7 (S7) reduced mutant htt by 40% and the wild typehuntingtin by 6%.

The inventors found that formulated LNP siRNAs were distributed broadlyfollowing intrastriatal infusion, that formulated LNP siRNA reduced Httin adult mouse brain at biologically relevant dose, and siRNAs targetingsequences targeting the expansion provided for allele specificsilencing.

The inventors also found that miRNA shuttles for allele specificsilencing of htt could effectively be used (FIG. 4). miRNA shuttlesbased on the siRNA sequence 7 (S7) were generated. To assess silencespecificity, HEK293 cells were co-transfected with wild type and mutanthuntingtin plasmids and mi7A1, mi7A2, mi7B1, mi7B2 or miGFP as acontrol. Cells were harvested 24 hours after transfection and wild typeand mutant Htt silencing was determined by western blot. Mi7A1 and mi7A2had the most preferential silencing profile, the latter the mostbeneficial.

Sequence mi7A1 silences very efficiently either wild type or mutanthuntingtin. This is possibly due to an excess of mi7A1 production. Thespecificity of silencing of mi7A1 at high and low doses was compared.HEK293 cells were transfected with two different amounts of mi7A1 andprotein lysates were obtained 24 hours after transfection. Silencing ofboth wild type and mutant huntingtin was determined by western blot withspecific antibodies against the epitope tags (FIGS. 5A and 5B). Datashows that preferential silencing for the mutant huntingtin is achievedwhen mi7A1 is transfected at a low dose. FIG. 5A shows normal Htt, andFIG. 5B shows mutant Htt.

The inventors also evaluated the strand biasing of miR shuttles (FIG.6). Different mutations were introduced to the 3′ end of the sensestrand of the mi7 sequences (mi7A2 and mi7B2) to promote antisensestrand loading into the RISC. To determine which strand waspreferentially loaded several luciferase reporter constructs based onpsicheck2 vector were designed. HEK293 cells were cotransfected withboth mi7 shuttle and a reporter construct for each strand and 24 hourslater cell extracts were obtained. Sequences 7A1 and 7A2 showedexceptional strand biasing.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The terms “comprising,” “having,”“including,” and “containing” are to be construed as open-ended terms(i.e., meaning “including, but not limited to”) unless otherwise noted.Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

What is claimed is:
 1. An expression cassette comprising a nucleic acidencoding an miRNA comprising a stem sequence, a sense strand of 15 to 30nucleotides in length, a loop sequence of 4 to 50 nucleotides in length,an antisense strand 15 to 30 nucleotides in length, and a stem sequence,wherein the miRNA has at least 80% sequence identity compared tofull-length miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2(SEQ ID NO:8).
 2. The expression cassette of claim 1, further comprisinga promoter.
 3. The expression cassette of claim 2, wherein the promoteris a CMV, RSV, pol II or pol III promoter.
 4. The expression cassette ofclaim 1, further comprising a marker gene.
 5. A vector comprising theexpression cassette of claim
 1. 6. The vector of claim 5, wherein thevector is an adenoviral, lentiviral, adeno-associated viral (AAV),poliovirus, HSV, or murine Maloney-based viral vector.
 7. A cellcomprising: (a) an expression cassette comprising a nucleic acid havingat least 80% sequence identity compared to full-length miHD7A-2 (SEQ IDNO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8); or (b) avector comprising an expression cassette, wherein the expressioncassette comprises a nucleic acid encoding an miRNA comprising a stemsequence, a sense strand of 15 to 30 nucleotides in length, a loopsequence of 4 to 50 nucleotides in length, an antisense strand 15 to 30nucleotides in length, and a stem sequence, wherein the miRNA has atleast 80% sequence identity compared to full-length miHD7A-2 (SEQ IDNO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8).
 8. An isolatedor purified miRNA comprising a stem sequence, a sense strand of 15 to 30nucleotides in length, a loop sequence of 4 to 50 nucleotides in length,an antisense strand 15 to 30 nucleotides in length, and a stem sequence,wherein the miRNA has at least 80% sequence identity compared tofull-length miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2(SEQ ID NO:8).
 9. The vector of claim 5, wherein the vector is an AAVvector.
 10. An expression cassette comprising a nucleic acid encoding anmiRNA comprising a stem sequence, a sense strand of 15 to 30 nucleotidesin length, a loop sequence of 4 to 50 nucleotides in length, anantisense strand 15 to 30 nucleotides in length, and a stem sequence,wherein the miRNA is miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), ormiHD7B-2 (SEQ ID NO:8).
 11. An isolated or purified miRNA comprising astem sequence, a sense strand of 15 to 30 nucleotides in length, a loopsequence of 4 to 50 nucleotides in length, an antisense strand 15 to 30nucleotides in length, and a stem sequence, wherein the miRNA ismiHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ IDNO:8).
 12. The expression cassette of claim 1, wherein the miRNA has atleast 80% sequence identity compared to full-length miHD7A-2 (SEQ IDNO:6).
 13. The expression cassette of claim 1, wherein the miRNA has atleast 80% sequence identity compared to full-length miHD7B-1 (SEQ IDNO:7).
 14. The expression cassette of claim 1, wherein the miRNA has atleast 80% sequence identity compared to full-length miHD7B-2 (SEQ IDNO:8).
 15. The expression cassette of claim 10, wherein the miRNA ismiHD7A-2 (SEQ ID NO:6).
 16. The expression cassette of claim 10, whereinthe miRNA is miHD7B-1 (SEQ ID NO:7).
 17. The expression cassette ofclaim 10, wherein the miRNA is miHD7B-2 (SEQ ID NO:8).